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Berkeley MCELLBI 140 - Enhancing zinc-finger-nuclease activity

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Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architecturesRESULTSIsolation of cold-sensitive FokI mutantsDesign of new engineered FokI cleavage half-domainsEnhanced activity in primary cellsPreservation of ZFN specificityPortability of the new FokI mutationsGeneration of enhanced orthogonal ZFN pairsDISCUSSIONMethodsONLINE METHODSYeast reporter strain.Random mutagenesis.Gap repair and screen.Cell culture and transfection.ZFN reagents.Surveyor nuclease (Cel-1) assay.RFLP knock in assay.g-H2AX staining.Statistical testing.Gels and blots.Western blot.AcknowledgmentsAUTHOR CONTRIBUTIONSCOMPETING FINANCIAL INTERESTSReferencesFigure 1 Isolation of FokI cold-sensitive mutants.Figure 2 Isolated mutations cluster to the FokI dimer interface.Figure 3 Enhanced activity of the ELD:KKK and ELD:KKR architecture.Figure 4 Improved activity of new ZFN mutants in primary cells.Figure 5 Preservation of the obligate heterodimer specificity.Table 1 | Engineered cleavage domain nomenclature© 2010 Nature America, Inc. All rights reserved.ArticlesnAture methods | ADVANCE ONLINE PUBLICATION | 1Zinc-finger nucleases (ZFns) drive efficient genome editing by introducing a double-strand break into the targeted gene. cleavage is induced when two custom-designed ZFns heterodimerize upon binding dnA to form a catalytically active nuclease complex. the importance of this dimerization event for subsequent cleavage activity has stimulated efforts to engineer the nuclease interface to prevent undesired homodimerization. here we report the development and application of a yeast-based selection system designed to functionally interrogate the ZFn dimer interface. We identified critical residues involved in dimerization through the isolation of cold-sensitive nuclease domains. We used these residues to engineer ZFns that have superior cleavage activity while suppressing homodimerization. the improvements were portable to orthogonal domains, allowing the concomitant and independent cleavage of two loci using two different ZFn pairs. these ZFn architectures provide a general means for obtaining highly efficient and specific genome modification.Zinc-finger nucleases (ZFNs) are hybrid restriction enzymes composed of a customizable zinc-finger protein DNA-binding domain fused to the cleavage domain of the FokI endonuclease1. As the zinc-finger protein DNA-binding domain can be engi-neered to bind with high specificity to an investigator’s chosen sequence2, ZFNs enable a DNA cleavage event to be targeted to effectively any genomic location. Subsequent repair of the ZFN-induced double-strand break by the evolutionarily conserved non-homologous end-joining or homology-directed repair pathways facilitates the precise modification of the targeted endogenous gene. ZFN-driven ‘genome editing’ has been demonstrated in a broad range of species, including those for which facile reverse genetics was not available previously, and has been a highly effi-cient tool for making precise genomic modifications in trans-formed and primary human cells3,4.Given the broad potential utility of ZFNs in genome engi-neering, considerable effort has been directed to methods that increase the cleavage activity and/or specificity of ZFNs. Three major determinants have been examined to date; the zinc-finger protein DNA-binding domain5, the FokI nuclease domain6,7 and enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architecturesYannick Doyon, Thuy D Vo, Matthew C Mendel, Shon G Greenberg, Jianbin Wang, Danny F Xia, Jeffrey C Miller, Fyodor D Urnov, Philip D Gregory & Michael C Holmesthe linker sequence connecting the two8. Although optimization of the DNA-binding domain can improve a specific ZFN pair9, the modularity of the FokI domain offers the potential for improvements to be portable to all ZFNs. To this end, we and others previously used structure-guided design to develop modi-fied FokI domains that function as obligate heterodimers6,7 and thus increase specificity. However, the mutations originally described also exhibited a reduction in dimerization energy revealed as a reduced rate of cutting in vitro6 and in vivo6,10. As FokI dimerization is necessary for DNA cleavage11 the resi-dues important for dimer formation remain attractive targets for development of ZFN architectures with improved specificity and activity.Here we exploit a yeast-based selection system to interrogate the dimer interface. We adapted a reporter assay, previously developed for the identification of active nucleases from a panel of preassembled ZFNs12–15, for the isolation of mutations in the FokI domain conferring cold-sensitivity. We reasoned that the isolation of cold-sensitive mutants could pinpoint critical residues involved in dimerization because this class of mutations is often associated with defects in assembly of multisubunit protein complexes16. We report the identification of such mutations and their use to guide the rational design of new FokI domains that have superior cleavage activity and retain obligate heterodimer function. The engineered substitutions also increased the activity of recently described orthogonal FokI domains10, improving the efficiency and specificity with which independent loci can be concomitantly targeted using two ZFNs. These enhanced FokI domains were portable to many zinc-finger proteins, independent of cell type, and are a general solution for improved ZFN activity.resultsisolation of cold-sensitive Foki mutantsWe developed a selection system in Saccharomyces cerevisiae to isolate ZFN mutants displaying a cold-sensitivity phenotype, that is, cleavage activity that is severely diminished at lower temperatures but robust at higher ones. The system uses two inde-pendent single-strand annealing reporter constructs integrated into the yeast genome that contain a binding site for the CCR5-L ZFN homodimer17 (Fig. 1a). For both reporters, a ZFN-induced Sangamo BioSciences, Richmond, California, USA. Correspondence should be addressed to Y.D. ([email protected]).Received 4 June; accepted 5 novembeR; published online 5 decembeR 2010; doi:10.1038/nmeth.1539© 2010 Nature America, Inc. All rights reserved.2 | ADVANCE ONLINE PUBLICATION | nAture methodsArticlesdouble-strand break resulted in restoration of reporter gene expression (MEL1 or PHO5) and


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Berkeley MCELLBI 140 - Enhancing zinc-finger-nuclease activity

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