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Updated: Nov 20, 2025
Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins
Published on: June 14, 2018
Drug target validation in primary human natural killer cells using CRISPR RNP
Jai Rautela1,2, Elliot Surgenor1, Nicholas D Huntington1,2
1Molecular Immunology Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia.
Abstract:
The ability to genetically modify CD8 T cells using viral gene delivery has facilitated the development of next generation of cancer immunotherapies such as chimeric Ag receptor (CAR) T cells engineered to specifically kill tumor cells. Development of immunotherapies targeting NK cells have stalled in part by their resistance to traditional viral gene delivery systems. Here, an efficient approach is described to genetically edit human NK cells by electroporation and CRISPR-Cas9 ribonucleoprotein (RNP) complexes. Electroporation pulse codes and buffer optimization for protein uptake by human NK cells and viability, and the efficiency of this approach over other methods are detailed. To highlight the transformative step this technique will have for NK cell immunotherapy drug discovery, NCR1 and CISH are deleted in primary human NK cells and murine findings are validated on their key roles in regulating NK cell antitumor function.
Insights
Researchers developed an efficient CRISPR-Cas9 gene editing method for human NK cells, overcoming viral delivery challenges. This breakthrough advances NK cell immunotherapy for cancer treatment.
Area of Science:
- Immunology
- Molecular Biology
- Cancer Research
Background:
- Chimeric antigen receptor (CAR) T cell therapy has advanced cancer immunotherapy.
- Developing NK (natural killer) cell immunotherapies is hindered by resistance to viral gene delivery.
Purpose of the Study:
- To establish an efficient gene editing technique for human NK cells.
- To validate the role of specific gene deletions in NK cell antitumor activity.
Main Methods:
- Utilized electroporation and CRISPR-Cas9 ribonucleoprotein (RNP) complexes for genetic modification of human NK cells.
- Optimized electroporation parameters and buffers for enhanced protein delivery and cell viability.
- Deleted NCR1 and CISH genes in primary human NK cells.
Main Results:
- Demonstrated an efficient method for genetically editing human NK cells via electroporation and CRISPR-Cas9 RNP.
- Validated the crucial roles of NCR1 and CISH in regulating NK cell-mediated antitumor functions, consistent with murine models.
- Showcased the potential of this technique for advancing NK cell immunotherapy drug discovery.
Conclusions:
- Electroporation with CRISPR-Cas9 RNPs provides an efficient platform for human NK cell genetic engineering.
- This method overcomes limitations of viral gene delivery for NK cell-based therapies.
- The findings support the development of novel NK cell immunotherapies for cancer treatment.
