nature biotechnology volume 27 number 9 September 2009 851 plants and rats14���19, and has also been used to edit the CCR5 locus in hESCs13. We used the locus encoding the pluripotency-associated gene OCT4 (also known as POU5F1)���one of the few genes that has been successfully targeted in hESCs6���to compare the efficiency of ZFN-mediated gene targeting in hESCs with that of conventional homologous recombination. We designed four ZFN pairs that rec- ognize unique sequences in the first intron of the OCT4 gene (Fig. 1a, Supplementary Fig. 1 and Supplementary Table 1) and generated targeting donor constructs with short homology arms for the three most active ZFNs pairs. Correct targeting of these donor constructs containing a splice acceptor (SA) followed by an enhanced green fluo- rescent protein (eGFP)-2A-puromycin cassette (2A is a self-cleaving peptide sequence) results in the expression of two proteins���a fusion protein comprising the first 132 amino acids of human OCT4 fused to eGFP (OCT4EX1-eGFP) and puromycin N-acetyltransferase���both under the control of the endogenous OCT4 promoter. Southern blot analysis using external probes 3��� and 5��� to the donor homology regions and an internal probe against eGFP revealed that for ZFN pair #1, 40 out of 42 individual cell lines established from puromycin-resistant clones were correctly targeted (efficiency 94% Fig. 1b and Table 1). ZFN pair #2 had a correct targeting frequency of 36���53% (Table 1). Of the remaining clones, most were correctly targeted in the OCT4 locus but also carried additional nonhomologous integrations (Fig. 1b and Table 1). ZFN pair #3 had the lowest targeting efficiency and generated only a few puromycin-resistant clones, some of which were correctly targeted. OCT4EX1-eGFP-targeted cells maintained a pluripo- tent state, as indicated by the expression of the pluripotency markers OCT4, NANOG, SOX2, Tra-1-60 and SSEA4 (Fig. 1c) and their abil- ity to form cell types originating from all three developmental germ layers in teratoma-formation assays (Fig. 1d). We detected expression of the OCT4EX1-eGFP fusion protein in hESCs by western blotting with antibodies against OCT4 and eGFP (Fig. 1e). When targeted hESCs were differentiated into fibroblasts, no OCT4EX1-eGFP pro- tein was detected (Fig. 1e) and the cells regained puromycin sensitivity, demonstrating the validity of the reporter expression. Unexpectedly, clones targeted by ZFN pair #1 showed significantly lower OCT4EX1-eGFP protein Realizing the full potential of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) requires efficient methods for genetic modification. However, techniques to generate cell type���specific lineage reporters, as well as reliable tools to disrupt, repair or overexpress genes by gene targeting, are inefficient at best and thus are not routinely used. Here we report the highly efficient targeting of three genes in human pluripotent cells using zinc-finger nuclease (ZFN)���mediated genome editing. First, using ZFNs specific for the OCT4 (POU5F1) locus, we generated OCT4-eGFP reporter cells to monitor the pluripotent state of hESCs. Second, we inserted a transgene into the AAVS1 locus to generate a robust drug-inducible overexpression system in hESCs. Finally, we targeted the PITX3 gene, demonstrating that ZFNs can be used to generate reporter cells by targeting non-expressed genes in hESCs and hiPSCs. Gene targeting by homologous recombination in hESCs has proven difficult, and since the derivation of the first hESCs more than 10 years ago, only a few reports have described successful gene target- ing1���9. These studies illustrate the utility of genetically modifying hESCs by gene targeting, but a general approach to manipulate the hESC genome is still lacking. Recently, a technique based on the introduction of DNA double-strand breaks by site-specific ZFNs to facilitate homologous recombination has been used to target endogenous genes in human cells10,11. A ZFN is generated by fusing the FokI nuclease domain to a DNA recognition domain composed of engineered C2H2 zinc-finger motifs that specify the genomic DNA binding site for the chimeric protein (Fig. 1a). Upon binding of two such fusion proteins at adjacent genomic sites, the nuclease domains dimerize, become active and cut the genomic DNA. When a donor DNA that is homologous to the target on both sides of the double-strand break is provided, the genomic site can be repaired by homology-directed repair, allowing the incorporation of exog- enous sequences placed between the homologous regions12,13. This technique, also called ���genome editing���, has been applied in systems not easily amenable to genetic modifications, such as zebrafish, Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases Dirk Hockemeyer1,4, Frank Soldner1,4, Caroline Beard1, Qing Gao1, Maisam Mitalipova1, Russell C DeKelver2, George E Katibah2, Ranier Amora2, Elizabeth A Boydston2, Bryan Zeitler2, Xiangdong Meng2, Jeffrey C Miller2, Lei Zhang2, Edward J Rebar2, Philip D Gregory2, Fyodor D Urnov2 & Rudolf Jaenisch1,3 1The Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA. 3Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. 2Sangamo BioSciences, Inc., Richmond, California, USA. 4These authors contributed equally to this work. Correspondence should be addressed to R.J. (jaenisch@wi.mit.edu). Received 8 June accepted 10 August published online 13 August 2009 doi:10.1038/nbt.1562 le TT e RS �� 2009 Nature America, Inc. All rights reserved.

852 volume 27 number 9 September 2009 nature biotechnology Figure 1 Targeting of OCT4 in heSCs using ZFNs. (a) Schematic overview depicting the targeting strategy for the OCT4 locus. Red boxes, probes used for Southern blot analysis blue boxes, exons of OCT4 arrows, genomic site cut by the respective ZFN pair. Shown above is a schematic of the donor plasmid design. Donor plasmids were created corresponding to the cleavage location of the three ZFN pairs and carried roughly 700-bp regions of homology to the OCT4 sequence. SA-eGFP, splice acceptor���eGFP sequence 2A, self-cleaving peptide sequence PURO, puromycin resistance gene polyA, polyadenylation sequence. Inset at upper left is a cartoon of two ZFNs binding at a specific genomic site (yellow), leading to dimerization of the FokI nuclease domains. (b) Southern blot analysis of BG01 cells targeted with the indicated ZFN pairs using the corresponding donor plasmids. Genomic DNA was either digested with ecoRI and hybridized with the external 3��� probe or digested with SacI and hybridized with the external 5��� probe or internal eGFP probe. Correctly targeted clones without additional integrations are indicated in red. Fragment sizes: for 5��� probe and eGFP probe, wt, 6.4 kb, targeted, 8.4 kb for 3��� probe, wt, 7.1 kb, targeted, 9.1 kb. (c) Immunofluorescence staining of BG01 cells targeted with the indicated ZFN pairs using the corresponding donor plasmids. Cells were stained for the pluripotency markers OCT4, NANOG, SOX2, Tra-1-60 and SSeA4. (d) Hematoxylin and eosin staining of teratoma sections generated from BG01 cells targeted with the indicated ZFN pairs and the corresponding donor plasmids. (e) Western blot analysis for the expression of OCT4 and eGFP in BG01 wild-type (wt) cells and BG01 cells targeted with the indicated ZFN pairs using the corresponding donor plasmids. Cell extracts were derived from either undifferentiated cells (eS) or in vitro���differentiated fibroblast-like cells (Fib.) -OCT4 GFP -OCT4 GFP -OCT4 BG01 OCT4 ZFN#1-1 OCT4 ZFN#1-2 OCT4 ZFN#2-2 OCT4 ZFN#2-4 ES ES ES ES ES Fib. Fib. Fib. Fib. Fib. BG01 OCT4 ZFN#1-1 OCT4 ZFN#1-2 OCT4 ZFN#2-2 OCT4 ZFN#2-4 ES ES ES ES ES Fib. Fib. Fib. Fib. Fib. GFP wester n OCT4 wester n a b SA-GFP-PUR O control SA-GFP-PURO +OCT4 ZFN#2 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 - 9.4 kb -6.6 kb - 4.4 kb - 23kb - 9.4 kb - 6.6 kb - 4.4 kb - 23 kb - 9.4 kb - 6.6 kb - 4.4 kb 5��� probe 3��� probe GFP probe SA-GFP-PURO +OCT4 ZFN#1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 - 23 kb - 9.4 kb - 6.6 kb - 4.4 kb - 23 kb - 9.4 kb - 6.6 kb - 4.4 kb - 23 kb - 9.4 kb - 6.6 kb 5��� probe 3��� probe GFP probe wt- Target- wt- Target- Target- Ectoderm Mesoderm Endoderm Neuronal rosettes Pigm. neuroepithel Cartilage Bone Smooth muscle c d e Intestinal epithelium Intestinal epithelium Pigm. neuroepithel Cartilage, bone Neuronal rosettes OCT4-GFP ZFN#1 OCT4-GFP ZFN#2 OCT4 NANO G SO X2 TRA-1-60 SSEA4 OCT4-GFP ZFN#1 OCT4-GFP ZFN# 2 2 kb Ex1 2 3 4 5 SacI EcoRI SacI EcoRI Donor targeting vector OCT4-GFP1 Donor targeting vector OCT4-GFP2 ZFN pair 3 ZFN pair 1 ZFN pair 2 5��� arm 3��� arm SA-GFP PURO polyA 2A 5��� arm 3��� arm SA-GFP PURO polyA 2A 5��� probe 3��� probe OCT4 locus: C A G A C C T G G C A C C C A G G A G A G G A G C A G G C A G G G T C A G C T G T C T G G A C C G T G G G T C C T C T C C T C G T C C G T C C C A G T C G A 0.25 mm le TT e RS �� 2009 Nature America, Inc. All rights reserved.