Besides improvements in the delivery of nucleotides and cell culture conditions, quantum leaps of genetic modification efficiencies were achieved through the application of designer nucleases, including zinc finger nucleases, transcription activator-like effector nuclease and clustered regularly interspaced short palindromic repeat RNA-guided Cas nuclease in the last decade. We and others have shown efficient gene targeting in human embryonic stem cells and induced pluripotent stem cells using these designer nucleases. Aided by fluorescence activated cell sorting of transfected cells, TALENs and CRISPR/Cas were shown to mediate high-efficiency single-gene indel mutagenesis through the error-prone non-homologous end-joining mechanism and defined single-nucleotide alterations through homology-directed repair mechanism in human pluripotent stem cells. However, applications of human stem cells in imaging, drug-screening and gene therapy would prefer using cells bearing targeted insertion and stable expression of large DNA CT99021 fragments such as reporters or minigene cassettes. Therefore it��s also highly desirable to engineer multiple genes simultaneously in human pluripotent stem cells to save time and cost. The one-step generation of mouse ESCs and mice carrying multiple indels, and rat with multiple floxed alleles indicated it��s possible to do so with highly active designer nucleases. Safe-harbor loci, which permit robust expression of AZ 960 integrated transgenes in the mammalian genome, provide defined ��landing sites�� for large exogenous DNA, such as minigenes and reporter cassettes. Ideally, genomic safe-harbors are loci that not only enable adequate, stable expression of the integrated material, but also minimize impacts on any nearby endogenous gene structures or functions. Focusing on the safety concerns, one group has proposed criteria by which to evaluate potential safe-harbors based on known human genome information, including; a distance of at least 50kb from the 5�� end of any gene, at least 300kb from any cancerrelated gene and microRNA, and a location outside of transcriptional units and ultraconserved regions. While the proposed set of guidelines is helpful to identify potential safe-harbors, if strictly adhered to, it would exclude some widely studied and used safe-harbors, such as PPP1R12C/AAVS1 and hROSA26,which have been shown to allow robust transgene expression in engineered human pluripotent stem cells and their differentiated lineages. Therefore we have taken a different approach to identify a genomic safe-harbor that enables sustainable gene expression and can be efficiently and definitively targeted by designer nucleases to engineer various human cell types.