Generation of Reporter Human Pluripotent Stem Cells Using CRISPR/Cas9 Editing (original) (raw)

Abstract

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-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-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.

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Acknowledgments

We would like to express our sincere gratitude to Ms. Kumiko Omori for her administrative support. This research was supported in part by the grants from AMED (23bm1423010h0001) to YH and Uehara Memorial Foundation to YH.

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Authors and Affiliations

  1. iPS Cell Advanced Characterization and Development Team, RIKEN BioResource Research Center, Ibaraki, Japan
    Yohei Hayashi
  2. Gene Engineering Division, RIKEN BioResource Research Center, Ibaraki, Japan
    Koji Nakade

Authors

  1. Yohei Hayashi
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  2. Koji Nakade
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Corresponding author

Correspondence toYohei Hayashi .

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Editors and Affiliations

  1. Department of Biochemistry & Molecular Biology, Louisiana State University Health Sciences, Shreveport, LA, USA
    Baojin Ding
  2. Department of Geriatrics - Xiangya Hospital, Central South University, Changsha City, Hunan, China
    Yu Tang

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Hayashi, Y., Nakade, K. (2024). Generation of Reporter Human Pluripotent Stem Cells Using CRISPR/Cas9 Editing. In: Ding, B., Tang, Y. (eds) Human Induced Pluripotent Stem Cells. Neuromethods, vol 210. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3999-3\_7

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