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CRISPR/Cas9 Ribonucleoprotein Complex-Mediated Efficient B2M Knockout in Human Induced Pluripotent Stem Cells (iPSCs)

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Induced Pluripotent Stem (iPS) Cells

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2454))

Abstract

Advances in induced pluripotent stem cell (iPSC) technology provide a renewable source of cells for tissue regeneration and therefore hold great promise for cell replacement therapy. However, immune rejection of allograft due to human leukocyte antigen (HLA) mismatching remains a major challenge. Considerable efforts have been devoted to overcoming the immunogenicity of allograft transplantation. One of the approaches is an elimination of HLA molecules on the surface of allogeneic cells using genome editing technology to generate universal stem cells. Here, we present a simple and effective genome editing approach to knockout the β-2-immunoglobulin (B2M) gene, which encodes B2M protein that forms a heterodimer with HLA class I proteins, in induced pluripotent stem cells (iPSCs) leading to HLA class I (HLA-I) depletion. We also describe detailed procedures for validation of the B2M-knockout iPSCs using flow cytometry, and genotypic analysis for potential off-target regions. Our protocol is also applicable for knocking out other genes in iPSCs and other cell types.

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Acknowledgments

This study was supported by a grant from Thailand Research Fund (grant no. RSA6280090), Siriraj Research Fund, Faculty of Medicine Siriraj Hospital, Mahidol University (grant number (IO) R016333015), the National Research Council of Thailand (NRCT): NRCT-RGJ63012-126 and the Program Management Unit for Human Resources & Institutional Development, Research and Innovation (Grant No. B05F630080). NT is supported by Siriraj Graduate Scholarship, Faculty of Medicine Siriraj Hospital, Mahidol University. MW is supported by Chalermphrakiat Grant, Faculty of Medicine Siriraj Hospital, Mahidol University.

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Authors and Affiliations

  1. Siriraj Center for Regenerative Medicine, Research Department, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand

    Nontaphat Thongsin & Methichit Wattanapanitch

  2. Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand

    Nontaphat Thongsin

Authors
  1. Nontaphat Thongsin
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  2. Methichit Wattanapanitch
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Corresponding author

Correspondence to Methichit Wattanapanitch .

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Editors and Affiliations

  1. Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada

    Andras Nagy

  2. Ottawa, ON, Canada

    Kursad Turksen

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Thongsin, N., Wattanapanitch, M. (2021). CRISPR/Cas9 Ribonucleoprotein Complex-Mediated Efficient B2M Knockout in Human Induced Pluripotent Stem Cells (iPSCs). In: Nagy, A., Turksen, K. (eds) Induced Pluripotent Stem (iPS) Cells. Methods in Molecular Biology, vol 2454. Humana, New York, NY. https://doi.org/10.1007/7651_2021_352

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  • DOI: https://doi.org/10.1007/7651_2021_352

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2118-9

  • Online ISBN: 978-1-0716-2119-6

  • eBook Packages: Springer Protocols

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