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CRISPR/Cas9-Based Gene Engineering of Human Natural Killer Cells: Protocols for Knockout and Readouts to Evaluate Their Efficacy

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Innate Lymphoid Cells

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2121))

Abstract

Natural killer (NK) cells are cytotoxic lymphocytes of our immune system with the ability to identify and kill certain virally infected and tumor-transformed cells. During the past 15 years, it has become increasingly clear that NK cells are involved in tumor immune surveillance and that they can be utilized to treat cancer patients. However, their ability to induce durable responses in settings of adoptive cell therapy needs to be further improved. One possible approach is to genetically engineer NK cells to augment their cytotoxicity per se, but also their ability to persist in vivo and home to the tumor-bearing tissue. In recent years, investigators have explored the potential of viral transduction and mRNA electroporation to modify NK cells. Although these methods have generated promising data, they are associated with certain limitations. With the increasing advances in the CRISPR/Cas9 technology, investigators have now turned their attention toward using this technology with NK cells as an alternative method. In this book chapter, we introduce NK cells and provide an historical overview of techniques to genetically engineer lymphocytes. Further, we elucidate protocols for inducing double-strand breaks in NK cells via CRISPR/Cas9 together with readouts to address its efficacy and functional outcome. We also discuss the pros and cons of the described readouts. The overall aim of this book chapter is to help introduce the CRISPR/Cas9 technology to the broader audience of NK cell researchers.

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Acknowledgements

We would like to acknowledge Marios Dimitriou and Teresa Montera Blanco for support with establishing the ddPCR technology and the MedH Flow Cytometry core facility (Karolinska Institutet), supported by KI/SLL, for providing cell analysis services. This work has been supported by funding from Swedish Society for Medicine, Wallenberg Clinical Fellow, Swedish Cancer Foundation, Swedish Childhood Cancer Association, and the Swedish Research Council.

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Authors and Affiliations

  1. Center for Hematology and Regenerative Medicine, Department of Medicine, Huddinge, Karolinska Institutet, Stockholm, Sweden

    Mélanie Lambert, Caroline Leijonhufvud, Filip Segerberg & Mattias Carlsten

  2. Center for Cellular Immunotherapies, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA

    J. Joseph Melenhorst

  3. Institute for Immunology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA

    J. Joseph Melenhorst

  4. Department of Pathology and Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA

    J. Joseph Melenhorst

  5. Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, USA

    J. Joseph Melenhorst

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  1. Mélanie Lambert
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  2. Caroline Leijonhufvud
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  3. Filip Segerberg
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  4. J. Joseph Melenhorst
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  5. Mattias Carlsten
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Corresponding author

Correspondence to Mattias Carlsten .

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Editors and Affiliations

  1. Laboratory of T cell Regulation, Institute of Cellular Medicine, Newcastle University, Newcastle Upon Tyne, UK

    Shoba Amarnath

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Lambert, M., Leijonhufvud, C., Segerberg, F., Melenhorst, J.J., Carlsten, M. (2020). CRISPR/Cas9-Based Gene Engineering of Human Natural Killer Cells: Protocols for Knockout and Readouts to Evaluate Their Efficacy. In: Amarnath, S. (eds) Innate Lymphoid Cells . Methods in Molecular Biology, vol 2121. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0338-3_18

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  • DOI: https://doi.org/10.1007/978-1-0716-0338-3_18

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