Research abstract
Article abstract
Nature Biotechnology 25, 778 - 785 (2007)
Published online: 1 July 2007 | doi:10.1038/nbt1319
An improved zinc-finger nuclease architecture for highly specific genome editing
Jeffrey C Miller1, Michael C Holmes1, Jianbin Wang1, Dmitry Y Guschin1, Ya-Li Lee1, Igor Rupniewski1, Christian M Beausejour1,2, Adam J Waite1, Nathaniel S Wang1, Kenneth A Kim1, Philip D Gregory1, Carl O Pabo1,2 & Edward J Rebar1
Abstract
Genome editing driven by zinc-finger nucleases (ZFNs) yields high gene-modification efficiencies (>10%) by introducing a recombinogenic double-strand break into the targeted gene. The cleavage event is induced using two custom-designed ZFNs that heterodimerize upon binding DNA to form a catalytically active nuclease complex. Using the current ZFN architecture, however, cleavage-competent homodimers may also form that can limit safety or efficacy via off-target cleavage. Here we develop an improved ZFN architecture that eliminates this problem. Using structure-based design, we engineer two variant ZFNs that efficiently cleave DNA only when paired as a heterodimer. These ZFNs modify a native endogenous locus as efficiently as the parental architecture, but with a >40-fold reduction in homodimer function and much lower levels of genome-wide cleavage. This architecture provides a general means for improving the specificity of ZFNs as gene modification reagents.
- Sangamo BioSciences, Inc., Pt. Richmond Tech Center, 501 Canal Blvd., Suite A100 Richmond, California 94804, USA.
- Present addresses: Département de pharmacologie, Centre de Recherche, CHU Ste-Justine 3175, Côte Ste-Catherine, Montréal, Quebec H3T 1C5, Canada (C.M.B.) and Department of Systems Biology, Harvard Medical School, 200 Longwood Avenue, WAB 536, Boston, Massachusetts 02115, USA (C.O.P.).
Correspondence to: Edward J Rebar1 e-mail: erebar@sangamo.com
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