New IO Paper Alert: CRISPR Screens Uncover New Targets to Boost CAR-NK Cell Therapy

CRISPR-based screening is opening new avenues for advancing adoptive natural killer (NK) cell therapy, which, when engineered with chimeric antigen receptors (CARs), has demonstrated safety and efficacy in hematologic malignancies but continues to face major limitations in solid tumors due to poor persistence, functional exhaustion, and tumor microenvironment–driven suppression. Overcoming these obstacles requires a systematic understanding of the genetic and molecular programs that regulate NK cell fitness.

In this study, researchers developed PreCiSE, the first genome-wide CRISPR screening platform optimized for primary human NK cells, enabling unbiased interrogation of more than 19,000 genes under clinically relevant conditions such as repeated tumor exposure, TGFβ signaling, lactic acid–induced acidity, hypoxia, and manufacturing stress. Using this approach, the team identified novel regulators, including MED12, ARIH2, and CCNC, whose disruption reprograms NK cells for enhanced persistence, cytotoxicity, and resistance to immunosuppression, providing a roadmap for next-generation CAR-NK therapies.

Title: Genome-wide CRISPR screens identify critical targets to enhance CAR-NK cell antitumor potency

Authors: Alexander Biederstädt1, Rafet Basar1, Jeong-Min Park, Nadima Uprety, Rejeena Shrestha,  Francia Reyes Silva,  Merve Dede,  John Watts,  Sunil Acharya, Donghai Xiong,  Bin Liu, May Daher, Hind Rafei, Pinaki Banerjee, Ping Li, Sanjida Islam, Huihui Fan, Mayra Shanley, Jingling Jin,  Bijender Kumar, Vernikka Woods, Paul Lin, Silvia Tiberti, Ana Karen Nunez Cortes, Xin Ru Jiang, Inci Biederstädt, Patrick Zhang, Ye Li, Seema Rawal, Enli Liu, Luis Muniz-Feliciano, Gary M. Deyter, Elizabeth J. Shpall, Natalie Wall Fowlkes,  Ken Chen, Katayoun Rezvani.

Published in Cancer Cell, August 2025

Background

Allogeneic natural killer (NK) cells engineered with chimeric antigen receptors (CARs) have demonstrated safety and activity in hematologic cancers. However, their clinical efficacy in solid tumors is limited by poor persistence, functional exhaustion, and immunosuppressive tumor microenvironments (TME). To overcome these barriers, researchers at MD Anderson Cancer Center developed a large-scale CRISPR screening platform to systematically identify genetic regulators of NK cell function.

Methods and Study Design

The team created PreCiSE, a genome-wide CRISPR discovery system optimized for primary human NK cells. Cord blood–derived NK cells were edited using pooled retroviral sgRNA libraries (77,736 guides targeting 19,281 genes) combined with Cas9 electroporation. Screens were performed under multiple clinically relevant stress conditions, including repeated pancreatic tumor re-challenges, TGFβ exposure, hypoxia (4% O₂), lactic acid, and prolonged ex vivo expansion.

Primary human NK cells were isolated from cord blood, expanded with engineered feeder cells and IL-2, and subjected to pooled CRISPR knockout library editing. To ensure high-quality coverage, NK cells were transduced at a low multiplicity of infection (MOI 0.3), which enabled single-guide integration, followed by puromycin selection to enrich for edited cells. The experimental framework included two complementary approaches: a transcription factor–focused screen covering 1,632 genes with 11,364 guides, and a genome-wide screen targeting 19,281 genes with more than 77,000 sgRNAs.

To model clinically relevant barriers, the edited NK cells were studied under several conditions. Repeated tumor re-challenge assays were performed using pancreatic cancer cells, including Panc1, PATC148, and Capan-1, to induce progressive NK exhaustion. Functional degranulation assays were used to separate NK cells with high versus low CD107a expression after tumor contact. Immunosuppressive microenvironmental stress was modeled by exposing NK cells to TGFβ, lactic acid, ammonium chloride, itaconic acid, hypoxia at 4% oxygen, and dexamethasone. In parallel, screens were conducted during prolonged ex vivo expansion to simulate the stresses of therapeutic NK cell manufacturing.

Outcomes were assessed by next-generation sequencing of sgRNA abundance to identify enriched or depleted targets, multiparameter flow cytometry and mass cytometry to define functional NK subsets, Seahorse assays to quantify metabolic reprogramming, and single-cell RNA sequencing to capture transcriptional shifts. Finally, functional validation was performed in vitro through serial tumor-killing and 3D spheroid assays, and in vivo using orthotopic pancreatic cancer and systemic multiple myeloma mouse models, providing a comprehensive readout of how specific genetic perturbations influenced NK cell persistence, cytotoxicity, and resistance to immunosuppression.

Results

• Efficient editing was achieved, with >90% loss of CD45 expression confirming knockout success.
• Transcription factor screens identified PRDM1 (Blimp-1) and RUNX3 as recurrent suppressors of NK function, both elevated in tumor-infiltrating NK cells from pancreatic cancer patients.
• Genome-wide screens revealed MED12, ARIH2, CCNC, PTEN, and CISH as key regulators.
• Deletion of MED12 significantly improved NK cytotoxicity against resistant tumors (PATC148, MM1S, THP-1, MOLM-14) and enhanced activity of IL-15–armored CAR-NK cells targeting TROP2 and CD70.
• Dual knockout of ARIH2 and CCNC reprogrammed NK cells toward a metabolically fit, highly activated phenotype, with increased oxygen consumption and pro-inflammatory cytokine secretion.
• In vivo, ARIH2/CCNC knockout CAR-NK cells improved infiltration, tumor clearance, and survival in orthotopic pancreatic cancer and systemic multiple myeloma mouse models.
• Integrated analysis identified conserved pathways involving apoptosis (FAS, CASP3), NF-κB regulation (NFKBIA), and ubiquitination (USP22, UBE2E1), shared with T-cell CRISPR screen datasets.

Key Findings

• Top targets: MED12, ARIH2, CCNC (novel regulators enhancing NK potency).
• NK cells with ARIH2/CCNC KO showed superior proliferation, IL-15 responsiveness, and IFNγ signaling compared to single edits.Tumor suppressor pathways (PTEN, TP53 regulators) emerged as actionable levers for NK metabolic fitness.
• Screens revealed hypoxia resistance genes (CASP3, FASLG, ITGB2, LOX, CITED2) and TGFβ signaling modulators (SMAD2, SMAD4) as engineering targets.

Key Takeaway Messages

• This is the first genome-wide CRISPR screening atlas in human NK cells, covering >19,000 genes.
• Identified regulators (MED12, ARIH2, CCNC, CISH, SMAD2/4) provide rational gene-editing targets to overcome TME pressures.
• Multiplex editing strategies can reprogram NK cells for enhanced persistence, cytotoxicity, and metabolic fitness in solid tumors.
• Findings bridge NK and T cell biology, suggesting shared regulatory hubs (Mediator, CRL, SAGA complexes).

Conclusion

By developing a scalable CRISPR platform, researchers mapped conserved genetic networks driving NK cell dysfunction and identified actionable targets to enhance CAR-NK therapy. Disruption of MED12, ARIH2, and CCNC substantially improved NK cytotoxicity, persistence, and in vivo tumor clearance. These insights set the stage for next-generation NK cell therapies tailored to resist TME suppression and broaden efficacy beyond hematologic malignancies into solid tumors.

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