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Updated: Dec 18, 2025
Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins
Published on: June 14, 2018
Enhanced NK-92 Cytotoxicity by CRISPR Genome Engineering Using Cas9 Ribonucleoproteins
Rih-Sheng Huang1, Hsin-An Shih1,2, Min-Chi Lai1,2
1Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.
Abstract:
Natural killer (NK) cells are an attractive cell-type for adoptive immunotherapy, but challenges in preparation of therapeutic primary NK cells restrict patient accessibility to NK cell immunotherapy. NK-92 is a well-characterized human NK cell line that has demonstrated promising anti-cancer activities in clinical trials. Unlimited proliferation of NK-92 cells provides a consistent supply of cells for the administration and development of NK cell immunotherapy. However, the clinical efficacy of NK-92 cells has not reached its full potential due to reduced immune functions as compared to primary NK cells. Improvements of NK-92 functions currently rely on conventional transgene delivery by mRNA, plasmid and viral vector with limited efficiencies. To enable precise genetic modifications, we have established a robust CRISPR genome engineering platform for NK-92 based on the nucleofection of Cas9 ribonucleoprotein. To demonstrate the versatility of the platform, we have performed cell-based screening of Cas9 guide RNA, multiplex gene knockout of activating and inhibitory receptors, knock-in of a fluorescent gene, and promoter insertion to reactivate endogenous CD16 and DNAM-1. The CRISPR-engineered NK-92 demonstrated markedly enhanced cytotoxicity and could mediate antibody-dependent cellular cytotoxicity against hard to kill cancer cell lines. Our genome editing platform is straightforward and robust for both functional studies and therapeutic engineering of NK-92 cells.
Insights
A new CRISPR genome engineering platform enhances Natural Killer (NK) cell line (NK-92) functions. This platform improves NK-92 cell-based immunotherapy by boosting anti-cancer activity and overcoming limitations of primary NK cell preparation.
Area of Science:
- Immunology
- Cell Biology
- Genetic Engineering
Background:
- Natural killer (NK) cells are crucial for adoptive immunotherapy, but primary NK cell preparation is challenging.
- The NK-92 cell line offers a consistent supply for immunotherapy but has suboptimal immune functions compared to primary NK cells.
- Current methods for improving NK-92 cell function via transgene delivery have limited efficiency.
Purpose of the Study:
- To establish a robust CRISPR genome engineering platform for the NK-92 cell line.
- To demonstrate the platform's versatility for genetic modification and functional enhancement of NK-92 cells.
- To improve NK-92 cell-mediated anti-cancer cytotoxicity for enhanced immunotherapy.
Main Methods:
- Developed a CRISPR-Cas9 ribonucleoprotein nucleofection platform for NK-92 cells.
- Performed cell-based screening of guide RNAs for precise gene editing.
- Executed multiplex gene knockout of key receptors and knock-in of a fluorescent reporter gene.
- Reactivated endogenous genes (CD16, DNAM-1) via promoter insertion.
Main Results:
- Engineered NK-92 cells exhibited significantly enhanced cytotoxicity against cancer cell lines.
- Demonstrated improved antibody-dependent cellular cytotoxicity (ADCC) in engineered NK-92 cells.
- Successfully modified NK-92 cells for potential therapeutic applications.
Conclusions:
- The established CRISPR platform is a straightforward and robust tool for NK-92 cell engineering.
- This platform enables precise genetic modifications to enhance NK cell functions for immunotherapy.
- CRISPR-engineered NK-92 cells show promise for overcoming limitations in current NK cell-based cancer therapies.
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