br Experimental Procedures br Acknowledgments br Introductio

Experimental Procedures
Acknowledgments
Introduction Clearly, conventional viral and nonviral gene transfer technologies resulting in the random integration of the introduced genetic elements and more or less unpredictable integration-site-dependent expression of the transgene are not in accordance with the requirements of current biomedical research. It has also been shown in animal experiments and clinical studies that random integration and insertional mutagenesis can result in the malignant transformation of stem cell transplants (Hacein-Bey-Abina et al., 2003; Modlich et al., 2009; Stein et al., 2010). It is therefore of the utmost importance to develop more precise techniques that enable efficient site-specific gene editing and safe long-term transgene expression at well-defined genomic integration sites in human PSCs (hPSCs) and especially iPSCs. In murine embryonic stem Sunitinib manufacturer (mESCs), gene targeting through homologous recombination (HR) has been utilized over the last 25 years to generate thousands of knockout mice, which has led to major advances in our basic understanding of mammalian biology, gene function, and disease mechanisms. Although the frequencies of HR are rather low in classical approaches (10−4 to 10−6 in mESCs) (Doetschman et al., 1988; Reid et al., 1991), such techniques have so far represented the standard approach for producing gene knockouts in mESCs and mice due to the relative robustness of mESC culture and high transfection rates in ESCs. Although two papers reported frequencies of HR (1.5–4 × 10−6) in a range similar to that seen in mESCs (Di Domenico et al., 2008; Zwaka and Thomson, 2003), conventional gene targeting in human ESCs (hESCs) is still considered to be more difficult and less successful due to challenging culture characteristics and lower transfection rates (Elliott et al., 2011; Goulburn et al., 2011; Irion et al., 2007). Moreover, until recently, the very low survival rates obtained after dissociation prevented fluorescence-activated cell sorting (FACS) and single-cell cloning. It is only since the invention of the Rho-associated coiled-coil kinase (ROCK) inhibitor Y-27632 that such techniques have become feasible for hPSCs (Zweigerdt et al., 2011). More recently, however, it has been demonstrated that targeted induction of double-strand breaks (DSBs) by employing tailored designer nucleases, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided nucleases greatly enhances HR (Fu et al., 2013; Mussolino and Cathomen, 2012; Rahman et al., 2011). ZFNs and TALENs consist of a target-specific DNA-binding domain fused to an unspecific nuclease domain, which induces a DSB upon activation. A ZFN/TALEN-induced DSB can be repaired either by nonhomologous end joining (NHEJ) or by HR (Shrivastav et al., 2008). Recent reports demonstrated that ZFNs and TALENs allow for not only efficient gene inactivation through NHEJ but also enhanced HR-based gene targeting in hPSCs (Hockemeyer et al., 2009, 2011; Soldner et al., 2011; Zou et al., 2009). Remarkably, ZFN/TALEN-based HR has already been applied for functional correction of genetic diseases either by genotypic correction of the defective gene (Yusa et al., 2011) or by insertion of the functional gene into a safe harbor locus (Zou et al., 2011). The majority of gene-targeting studies in hPSCs directly applied a transgene-based antibiotic selection of targeted clones (Hockemeyer et al., 2009, 2011; Sebastiano et al., 2011; Yusa et al., 2011; Zou et al., 2011). Clearly, further improvements in targeting efficiencies would not only minimize the required screening procedures but would considerably facilitate selection-independent targeting approaches in PSCs, including footprintless restoration of wild-type sequences in disease-specific iPSCs prior to their clinical application.