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

  • Mélanie Lambert
  • Caroline Leijonhufvud
  • Filip Segerberg
  • J. Joseph Melenhorst
  • Mattias CarlstenEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2121)


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.

Key words

NK cells Gene editing CRISPR/Cas9 Flow cytometry RT-PCR DNA sequencing Digital droplet PCR 



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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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Mélanie Lambert
    • 1
  • Caroline Leijonhufvud
    • 1
  • Filip Segerberg
    • 1
  • J. Joseph Melenhorst
    • 2
    • 3
    • 4
    • 5
  • Mattias Carlsten
    • 1
    Email author
  1. 1.Center for Hematology and Regenerative Medicine, Department of Medicine, HuddingeKarolinska InstitutetStockholmSweden
  2. 2.Center for Cellular ImmunotherapiesPerelman School of Medicine at the University of PennsylvaniaPhiladelphiaUSA
  3. 3.Institute for ImmunologyPerelman School of Medicine at the University of PennsylvaniaPhiladelphiaUSA
  4. 4.Department of Pathology and Laboratory MedicinePerelman School of Medicine at the University of PennsylvaniaPhiladelphiaUSA
  5. 5.Abramson Cancer CenterUniversity of PennsylvaniaPhiladelphiaUSA

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