Articles Enhancing zinc finger nuclease activity with improved obligate heterodimeric architectures 2010 Nature America Inc All rights reserved Yannick 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 Holmes Zinc 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 engineered 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 ZFNinduced double strand break by the evolutionarily conserved nonhomologous 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 efficient tool for making precise genomic modifications in transformed and primary human cells3 4 Given the broad potential utility of ZFNs in genome engineering 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 the 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 modified FokI domains that function as obligate hetero dimers6 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 residues 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 acti vity 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 RESULTS Isolation of cold sensitive FokI mutants We 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 independent 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 ydoyon sangamo com Received 4 June accepted 5 November published online 5 december 2010 doi 10 1038 nmeth 1539 nature methods ADVANCE ONLINE PUBLICATION Articles T 6L 48 d 2010 Nature America Inc All rights reserved K 49 44 6D 8M H Q486L 5 E K5 37L 48 Q486L K448M 59 A 4V T 48 L5 2T N496D E484V 6 H537L A482T Q Q 3M 53 5 K559T L563M 1R 31R N Q531R K4 50 V5 27 0S 12 Q531R V512M N M N K4 M 50 57 02R N500S K402R 0S 8S K427M N578S K4 69 N500S K469M N M 47 N476D 6D N476K N 47 6K G474S G 47 G474A 4S G D467E 47 4A D 46 7E HIS3 Mutagenized Fokl Q 6L CYC1t N Fokl I538T CCR5 L 48 FokI Chromosome IV MEL1 Q SSA PGK1 I538F GAL1 F 0 38 CCR5 L URA3 kanMX 20 10 HIS3 Chromosome IV EL1 30 l5 CCR5 L CCR5 L 40 I499T FokI CYC1t 50 T MEL FokI PGK1 CCR5 L natMX Fokl GAL1 38 PHO CCR5 L Chromosome II HO5 60 99 SSA FokI 22 C 30 C 37 C 70 l4 PGK1 c Fokl Mutagenic PCR l5 b PGK1 Chromosome II PHO5 Relative activity of wt a Figure 1 Isolation of FokI cold sensitive mutants a Schematic of two independent single strand annealing SSA reporter constructs containing a homodimer site for Anti Flag the CCR5 L ZFN that were integrated in the yeast genome The PHO5 SSA reporter contains the positive selection cassette natMX conferring dominant resistance to nourseothricin The MEL1 SSA reporter contains the URA3 gene for negative selection Anti H3 using 5 fluoroorotic acid 5 FOA in addition to the kanMX cassette conferring dominant resistance to geneticin G418 A DNA double stranded break induced by a functional homodimeric ZFN will result in reconstitution of the reporter genes and elimination
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