2007 Nature Publishing Group http www nature com naturebiotechnology ARTICLES 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 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 Zinc finger nucleases ZFNs are rapidly emerging as versatile reagents for gene modification These hybrid restriction enzymes which link the cleavage domain of FokI to a designed zinc finger protein ZFP 1 2 may be used to introduce a variety of custom alterations into the genomes of eukaryotic cells Examples range from precise sequence edits3 6 to the targeted integration of entire genes7 ZFNs initiate these events by introducing a double strand break at the site chosen for modification If an exogenous repair template is also supplied then sequence alterations encoded in this donor may be incorporated into the genome by homology directed repair HDR 8 Targeting of the cleavage event which is central to ZFN specificity and versatility is mediated by the ZFP domain This DNA binding domain has been characterized in great detail9 11 and may be engineered to recognize a wide variety of chosen DNA sequences12 16 ZFNs may offer a general method for engineering the genomes of diverse species as the requisite DNA repair pathways are highly conserved17 ZFN stimulated gene modification has been demonstrated in plants18 19 insects4 6 20 roundworms21 and human cells3 5 with endogenous gene correction rates as high as 18 5 Moreover donorfree delivery of ZFNs may be used for the targeted disruption of endogenous genes4 6 20 The generality of ZFN mediated gene modification raises the prospect of applications in diverse fields including crop engineering therapeutic gene correction cell line customization for biologics production and the development of nonmurine models of human disease Realizing the full potential of these approaches however will require the development of ZFN architectures and design strategies that yield efficient cleavage while minimizing off target effects An important feature of the current ZFN architecture is that DNA cleavage requires nuclease dimerization ZFNs as well as wild type FokI interact through their cleavage domains and are inactive as monomers22 25 As a consequence cleavage of a typical target requires the design of two different ZFNs for binding to adjacent half sites Fig 1a b Moreover the dimerization interaction is quite weak FokI remains monomeric at concentrations of at least 15 mM and dimerizes only when bound to its specific target23 26 From the standpoint of targeting specificity an advantage of this arrangement is that cleavage requires simultaneous binding of both ZFNs to their respective halfsites This has enabled the construction of ZFN dimers with cleavage specificities of up to 24 bp5 a length that exceeds the target size of other proposed gene modification agents such as the meganuclease ISce I27 and that offers the prospect for unique targeting within the human genome The development of ZFNs that specifically cleave even longer targets may be anticipated as individual ZFPs have been successfully designed for sequences as long as 18 bp28 31 Although the requirement for dimerization opens up the possibility of restricting cleavage to very long and rare sequences it also introduces a problem arising from the fact that protein protein interactions mediated by the wild type FokI cleavage domain are not themselves selective for the heterodimer species Fig 1c As a consequence the expression of any ZFN heterodimer e g Lwt Rwt in Fig 1c also yields two side product homodimers Lwt Lwt and Rwt Rwt that will be irrelevant for gene modification but may nonetheless limit safety or effectiveness by cleaving off target sequences Moreover if one ZFN is less specific than the other the 1Sangamo BioSciences Inc Pt Richmond Tech Center 501 Canal Blvd Suite A100 Richmond California 94804 USA 2Present addresses De partement de pharmacologie Centre de Recherche CHU Ste Justine 3175 Co te Ste Catherine Montre al 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 and requests for materials should be addressed to E J R erebar sangamo com Received 31 January accepted 4 June published online 1 July 2007 doi 10 1038 nbt1319 778 VOLUME 25 NUMBER 7 JULY 2007 NATURE BIOTECHNOLOGY ARTICLES Figure 1 DNA recognition and cleavage by zinc finger nucleases ZFNs wt wt a Sketch of a ZFN dimer bound to a typical nonpalindromic DNA target Rwt Right ZFP Lwt Cleavage Each ZFN consists of the cleavage domain of FokI fused to a zinc finger domain protein ZFP that has been customized to specifically recognize either a Fokl wt wt Lwt left or right half site indicated by blue and red boxes which are Lwt separated by a spacer of either 5 or 6 bp Simultaneous binding by both ZFNs enables dimerization of the FokI nuclease domain and DNA cleavage wt wt Rwt Note that endogenously active ZFNs have been created using four fingers Rwt Cleavage indicated here that each bind 12 bp sites5 as well as three fingers that Left ZFP domain each bind 9 bp sites4 6 20 21 b Three dimensional model corresponding to the arrangement in a DNA is shown in gray ZFNs are colored blue left ZFN or red right ZFN and reflective spheres denote zinc ions Zinc finger R helices that mediate sequence recognition
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