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hWe report here homologous recombination (HR)-mediated gene targeting of two different genes in human iPS cells (hiPSCs) and human ES cells (hESCs). HR-mediated correction of a chromoso-mally integrated mutant GFP reporter gene reaches efficiencies of 0.14%–0.24 % in both cell types trans-fected by donor DNA with plasmids expressing zinc finger nucleases (ZFNs). Engineered ZFNs that in-duce a sequence-specific double-strand break in the GFP gene enhanced HR-mediated correction by> 1400-fold without detectable alterations in stem cell karyotypes or pluripotency. Efficient HR-medi-ated insertional mutagenesis was also achieved at the endogenous PIG-A locus, with a> 200-fold enhancement by ZFNs targeted to that gene. Clonal PIG-A null hESCs and iPSCs with normal karyotypes were readily obtained. The phenotypic and biological defects were rescued by PIG-A transgene expres-sion. Our study provides the first demonstration of HR-mediated gene targeting in hiPSCs and shows the power of ZFNs for inducing specific genetic modifications in hiPSCs, as well as hESCs. exhibit unique characteristics similar to hESCs (Lowry et al 2008; Mali et al., 2008; Park et al., 2008; Takahashi et al 2007; Yu et al., 2007). Patient-specific iPSCs hold enormou promise for personalized cell replacement therapy, as well a for research of various human diseases. Full utilization of thes human pluripotent stem cells (hPSCs) will require the develop ment of efficient methods for performing gene targeting, sequence-specific and permanent genome modification tha exploits the cell’s ability to perform homologous recombinatio (HR). Gene targeting by HR has played a critical role in geneti studies of various systems, including the generation of kno ckout/knockin transgenic mouse models using mouse ESCs The efficiency of HR-mediated gene targeting in hESCs, ho wever, remains low even 10 years after its first report (Thomso et al., 1998), with only few successful studies published to dat using the method that is routinely performed in mouse ES