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Updated: Sep 10, 2025
Performing an In Vitro Genome-Wide CRISPR Knockout Screen in Chimeric Antigen Receptor T Cells
Published on: January 31, 2025
Genome-wide CRISPR screens identify critical targets to enhance CAR-NK cell antitumor potency
Alexander Biederstädt1, Rafet Basar2, Jeong-Min Park2
1Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Institute for Cell Therapy Discovery and Innovation, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Department of Medicine III: Hematology & Oncology, School of Medicine, Technical University of Munich, Munich, Germany.
Abstract:
Adoptive cell therapy using engineered natural killer (NK) cells is a promising approach for cancer treatment, with targeted gene editing offering the potential to further enhance their therapeutic efficacy. However, the spectrum of actionable genetic targets to overcome tumor and microenvironment-mediated immunosuppression remains largely unexplored. We performed multiple genome-wide CRISPR screens in primary human NK cells and identified critical checkpoints regulating resistance to immunosuppressive pressures. Ablation of MED12, ARIH2, and CCNC significantly improved NK cell antitumor activity against multiple treatment-refractory human cancers in vitro and in vivo. CRISPR editing augmented both innate and CAR-mediated NK cell function, associated with enhanced metabolic fitness, increased secretion of proinflammatory cytokines, and expansion of cytotoxic NK cell subsets. Through high-content genome-wide CRISPR screening in NK cells, this study reveals critical regulators of NK cell function and provides a valuable resource for engineering next-generation NK cell therapies with improved efficacy against cancer.
Insights
Engineered natural killer (NK) cells show promise for cancer therapy. Gene editing identified MED12, ARIH2, and CCNC as targets to enhance NK cell antitumor activity and overcome immunosuppression.
Area of Science:
- Immunology
- Genetics
- Oncology
Background:
- Adoptive cell therapy with engineered natural killer (NK) cells is a promising cancer treatment strategy.
- Targeted gene editing can enhance NK cell efficacy, but key genetic targets for overcoming tumor-induced immunosuppression are largely unknown.
Purpose of the Study:
- To identify critical genetic targets in human NK cells that regulate resistance to immunosuppressive pressures.
- To explore the potential of gene editing to improve NK cell-based cancer therapy.
Main Methods:
- Genome-wide CRISPR screens were performed in primary human NK cells.
- The functional impact of gene ablation on NK cell antitumor activity was assessed in vitro and in vivo against human cancers.
Main Results:
- Ablation of MED12, ARIH2, and CCNC significantly enhanced NK cell antitumor activity against treatment-refractory cancers.
- CRISPR editing improved both innate and chimeric antigen receptor (CAR)-mediated NK cell function.
- Editing led to enhanced metabolic fitness, increased pro-inflammatory cytokine secretion, and expansion of cytotoxic NK cell subsets.
Conclusions:
- This study reveals critical regulators of NK cell function and resistance to immunosuppression.
- The identified genes provide valuable targets for engineering next-generation NK cell therapies with improved efficacy for cancer treatment.
