Summary Custom-made zinc-finger nucleases (ZFNs) can induce targeted genome modifications with high efficiency in cell types including Drosophila, C. elegans, plants, and humans. A bottleneck in the application of ZFN technology has been the generation of highly specific engineered zinc-finger arrays. Here we describe OPEN (Oligomerized Pool ENgineering), a rapid, publicly available strategy for constructing multi-finger arrays, which we show is more effective than the previously published modular assembly method. We used OPEN to construct 37 highly active ZFN pairs which induced targeted alterations with high efficiencies (1 to 50%) at 11 different target sites located within three endogenous human genes (VEGF-A, HoxB13, CFTR), an endogenous plant gene (tobacco SuRA), and a chromosomally-integrated EGFP reporter gene. In summary, OPEN provides an “open-source” method for rapidly engineering highly active zinc-finger arrays, thereby enabling broader practice, development, and application of ZFN technology for biological research and gene therapy.

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ISG15 is an interferon-induced ubiquitin-like modifier which can be conjugated to distinct, but largely unknown, proteins. ISG15 has been implicated in a variety of biological activities, which encompass antiviral defense, immune responses, and pregnancy. Mice lacking UBP43 (USP18), the ISG15-deconjugating enzyme, develop a severe phenotype with brain injuries and lethal hypersensitivity to poly(I: Interferons (IFNs) are cytokines that communicate signals for a broad spectrum of cellular activities that encompass antiviral and immunomodulatory responses, as well as growth regulation. These pleiotropic cellular activities are mediated through a large number of proteins whose expression is triggered by activated interferon receptors present on almost all cells (3, 32). Intensive research established JAK/STAT as the principal intracellular signaling pathway downstream of interferon receptors (9,15,25). Despite great progress, our understanding of the complex IFN activities remains incomplete.CInterferon-stimulated gene 15/ubiquitin cross-reacting protein (designated ISG15/UCRP) is a 15-kDa ubiquitin-like protein identified as a product of an IFN-stimulated gene in humans (11). ISG15-homologous genes were found in several other species but are absent in yeast (26). ISG15 expression is induced in many cell types by IFNs, viral infection, bacterial endotoxins, double-stranded RNA, and genotoxic stress (7). Congruently, transcription factors of the interferon regulatory factor family (IRF) (IRF-1, IRF-3, IRF-4, IRF-7, and ICSBP/ IRF-8) that bind to the interferon-stimulated response element motif in the regulatory DNA region of ISG15, together with the ets factor PU.1, regulate ISG15 expression (28). ISG15 was also found to be strongly induced by NEMO/IB signaling (16).The mature ISG15 polypeptide is generated from a precursor by specific cleavage of the carboxyl-terminal extension (26), a feature common to several ubiquitin-like proteins. The ISG15 protein consists of two ubiquitin-like domains with an overall sequence similarity to ubiquitin of 59.3%. Moreover, the fold-determining sequences of ubiquitin are also very highly conserved in ISG15 (7). ISG15 contains the canonical LRGG motif at its C terminus, which is required for conjugation of ubiquitin and ubiquitin-like proteins to their targets. Similar to conjugation of ubiquitin and other ubiquitin-like molecules, such as SUMO or NEDD8, ISG15 is ligated by an isopeptide bond to several target proteins (17). UBE1L and UbcH8 were identified as E1-and E2-conjugating enzymes for ISG15, respectively (34,35). Recently, as a first protein substrate to which ISG15 is conjugated, serine-protease inhibitor (serpin 2a) was identified by mass spectrometry (8).The functional significance of the protein modification by ISG15 conjugation (ISGylation) is not yet known. However, the following observations strongly suggested that it may have important physiological activity. Conjugation of ISG15 to several cellular proteins increases rapidly after endotoxin (lipopolysaccharide [LP...

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Gene knockout in murine embryonic stem cells (ESCs) has been an invaluable tool to study gene function in vitro or to generate animal models with altered phenotypes. Gene targeting using standard techniques, however, is rather inefficient and typically does not exceed frequencies of 10−6. In consequence, the usage of complex positive/negative selection strategies to isolate targeted clones has been necessary. Here, we present a rapid single-step approach to generate a gene knockout in mouse ESCs using engineered zinc-finger nucleases (ZFNs). Upon transient expression of ZFNs, the target gene is cleaved by the designer nucleases and then repaired by non-homologous end-joining, an error-prone DNA repair process that introduces insertions/deletions at the break site and therefore leads to functional null mutations. To explore and quantify the potential of ZFNs to generate a gene knockout in pluripotent stem cells, we generated a mouse ESC line containing an X-chromosomally integrated EGFP marker gene. Applying optimized conditions, the EGFP locus was disrupted in up to 8% of ESCs after transfection of the ZFN expression vectors, thus obviating the need of selection markers to identify targeted cells, which may impede or complicate downstream applications. Both activity and ZFN-associated cytotoxicity was dependent on vector dose and the architecture of the nuclease domain. Importantly, teratoma formation assays of selected ESC clones confirmed that ZFN-treated ESCs maintained pluripotency. In conclusion, the described ZFN-based approach represents a fast strategy for generating gene knockouts in ESCs in a selection-independent fashion that should be easily transferrable to other pluripotent stem cells.