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TY - CHAP AU - Hayashi, Yohei AU - Nakade, Koji ED - Ding, Baojin ED - Tang, Yu PY - 2024 DA - 2024// TI - Generation of Reporter Human Pluripotent Stem Cells Using CRISPR/Cas9 Editing BT - Human Induced Pluripotent Stem Cells SP - 85 EP - 92 PB - Springer US CY - New York, NY AB - Differentiated cells derived from human pluripotent stem cells (hPSCs) have found wide applications in modeling cellular transplantation therapies, evaluating drug candidates, and advancing basic biology. The precision monitoring and selection of specific differentiated cell types can be achieved by knocking-in a cassette encoding a fluorescent protein at the end of various differentiation marker genes using genome-editing techniques. Although genome editing with homology-directed repair has been advanced to knock-in desired reporter genes into the human genome, the efficiency is still low due to the random insertion of donor vectors into the host genome. We established a method to use a “double-tk donor vector system” in which the expression units of the thymidine kinase of Herpes simplex virus (HSV-tkThymidine kinase of Herpes-simplex virus (HSV-tk)) are placed on both outer sides of homology arms. This system is useful for selecting knocked-in hPSCs compared to conventional donor vector systems with no or a single HSV-tkThymidine kinase of Herpes-simplex virus (HSV-tk) cassette. In this chapter, we describe this method for the generation of reporter hPSCs using CRISPR/Cas9 editing technology with this cellular suicide system based on double-tk donor vectors. SN - 978-1-0716-3999-3 UR - https://doi.org/10.1007/978-1-0716-3999-3\_7 DO - 10.1007/978-1-0716-3999-3_7 ID - Hayashi2024 ER -