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 Carlsten 
Source: Methods Mol Biol
Publication Date: (2020)
Issue: 2121: 213-219
Research Area:
Immunotherapy / Hematology
Cells used in publication:
Natural killer Cells (NK), human
Species: human
Tissue Origin: blood
Culture Media:
4D-Nucleofector™ X-Unit

1. Prepare the guide RNA (gRNA) by mixing Alt-R CRISPRCas9 crRNA and Alt-R CRISPR Cas9 tracrRNA in a sterile PCR tube at 1:1 ratio (1.25 µL of each) so the final concentration of the gRNA is 100 µM. Heat at 95 C for 5 min in a PCR thermal cycler. Cool down for 5–10 min to RT. Prepare the RNP complex by adding 1.5 µL of Cas9 and 1 µL of PBS to the gRNA and leave at RT for 10 min. Keep the RNP complex on ice (see Notes 1–3).
2. Turn on the 4D-Nucleofector™ System. Select the electroporation
protocol CM-137.
3. Determine the number of cells needed for the study, and collect the desired number of cells from the culture flask into a 50 mL tube.
4. Centrifuge the sample at 300  g for 7 min.
5. Aspirate the supernatant completely.
6. Wash the sample with 5 mL PBS; centrifuge at 300  g for 7 min.
7. Determine the density and total volume of cell suspension needed for transfection. Carefully suspend the cells in P3 buffer in 15 µL in a sterile PCR tube.
8. Without waiting, carefully add 5 µL of the RNP complex to the cells and mix carefully three times with the same pipette tip.
9. Transfer the contents of the PCR tube to a sterile cuvette in the Nucleocuvette Strip. Sweep each of the four corners of the chamber with cell suspension to make sure there are no air
bubbles left in the cuvette.
10. Put the lid on the strip and plug into the 4D-Nucleofector™ System.
11. Press “Start” and wait until the program indicates


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.