Jul 08, 2025

CAR-T and CAR-NK cell therapies have shown outstanding clinical performance against relapsed and refractory hematological malignancies. While both of these cell therapy modalities serve as powerful tools in the fight against cancer, they differ significantly in their biology and therapeutic properties. CAR-T cells are derived from a patient’s own T cells and require antigen-specific activation, often resulting in long-lived responses but also posing risks such as cytokine release syndrome (CRS) and graft-versus-host disease (GVHD). In contrast, CAR-NK cells originate from donor sources like cord blood or stem cells, can act without prior antigen priming, and are generally shorter-lived but safer, with minimal evidence of severe toxicities. Their ability to kill through antibody-dependent cellular cytotoxicity (ADCC) and innate receptors like NKG2D, without triggering GVHD, makes CAR-NK cells a promising "off-the-shelf" alternative to CAR-T therapies. Despite the success achieved in blood cancers, in the context of solid tumors, the efficacy of both CAR-T and CAR-NK cell therapies is limited by critical obstacles, such as their poor tumor infiltration capacity and susceptibility to inhibitory signals within the tumor microenvironment (TME).

Differences between CAR-T and CAR-NK cells

Table information retrieved from Table 1 of Chan et al. 2022.1 https://creativecommons.org/licenses/by/4.0/

Significant efforts have been made to identify immunosuppressive factors limiting the expansion, persistence, and cytotoxicity of CAR T cells. To date, much more is known about the signaling mechanisms reducing CAR-T cell function at the tumor site than those affecting CAR-NK cells.

Why are more immunomodulators known for T Cells than NK Cells?

CRISPR screens have been instrumental in identifying regulators of T cell function. Unsurprisingly, this strategy is now being implemented to learn more about the signals limiting NK cell anti-tumor function.

At AACR 2025, presentations by Dr. Alexander Biednerstadt and Dr. Marcella Mauss highlighted the impact of CRISPR/Cas9-based screening strategies in engineering more effective CAR-NK and CAR-T cell therapies.

Unlocking CAR-NK cells’ full potential with CRISPR screens

CAR-T cells have dominated the cancer cell therapy field. In contrast, NK cells, nature’s own cytotoxic defense squad, are beginning to step into the spotlight. But to truly compete, these cells must overcome some fundamental biological hurdles. That’s where CRISPR genome-wide screening has come in handy for Dr. Alexander Biedenkapp, who is now at the Technical University of Munich.

At AACR, Biedenkapp dove into his postdoctoral work in Dr. Katy Rezvani’s lab at MD Anderson, developing a genome-wide CRISPR screen in primary human NK cells.

CAR-NK cells offer a promising “off-the-shelf” immunotherapy option. Unlike autologous CAR-T therapies, NK cells don’t require patient matching and show minimal risk of graft-versus-host disease. Their safety profile is impressive, and early clinical trials have shown potential in treating hematologic cancers.

Despite these benefits and early promise, CAR-NK therapies struggle with three persistent problems, including limited in vivo persistence, functional exhaustion, and vulnerability to the tumor microenvironment (TME). Addressing these hurdles requires precise cellular reprogramming, and that's exactly what CRISPR screening enables.

To create opportunities for better NK cell therapies, Dr. Biedenkapp and colleagues built a powerful pooled CRISPR knockout screening platform tailored to primary human NK cells. The researchers implemented a strategy that involved the retroviral delivery of gRNA libraries into primary NK cells, followed by the electroporation of Cas9 protein to introduce gene knockouts. This was no small feat; CRISPR editing of NK cells has been challenging, but using electroporation of Cas9 can solve the issue of low expression efficiency.²

Next, they utilized tumor challenge models to apply selection pressure and employed functional readouts, such as degranulation (CD107a) and metabolic stress survival, to sort for high-performing NK cells.

Lastly, using next-generation sequencing (NGS) allowed them to identify which gene edits contributed to enhanced functionality. Essentially, this approach allowed them to track the edited NK cells that thrived under the most challenging tumor-like conditions.

As Dr. Biedenkapp shared, the screen yielded a rich atlas of both known and novel NK cell regulators critical in tuning their fitness, cytotoxicity, and persistence. From this massive dataset, three gene targets rose to the top, including MED12, ARHIH2, and CCNC.

They found that knocking out MED12, a component of the Mediator complex essential for transcriptional regulation, dramatically enhanced NK cell cytotoxicity. This effect was seen not only in unmodified NK cells but also in CAR-NKs targeting solid tumor antigens like TROP2 and CD70.

Similarly, knocking out ARIH2 or CCNC, previously unappreciated players in NK cell biology, served as potent enhancers of metabolic fitness. When combined, dual ARIH2/CCNC knockout CAR-NK cells showed the most potent improvements in engraftment, tumor infiltration, and cytotoxicity in a pancreatic cancer mouse model.

Overall, Dr. Biedenkapp’s CRISPR atlas is more than just a catalog; it’s a launchpad for building smarter, more resilient CAR-NK therapies.

CRISPR/Cas9-based in vivo screenings for next-generation CAR-T cell therapies

Dr. Mauss and collaborators at Massachusetts General Hospital in Boston, including CRISPR screening expert Dr. Rob Manguso, are taking bold steps to develop next-generation CAR-T cell therapies. The team has developed an in vivo CRISPR screening platform specifically for CAR-T cells.

Genome-wide CRISPR/Cas9 screens have been previously used in vitro to uncover genes controlling T cell function or exhaustion by applying different immunosuppressors.

However, Mauss and colleagues' method takes a step further by implementing this approach directly in animal models of cancer. About the selected approach, Dr. Maus shared, “We are not sure that in vitro stimulation necessarily is the best replicate of what happens in patients. And so we took the next best thing, which was to try to do this in vivo.”

This strategy's clear advantage is that it allows CAR-T cells to be exposed to real tumors and their complex microenvironment pressures, in the context of tumor-bearing immunodeficient mice.

Additionally, instead of performing a CRISPR genome-wide screening, the team chose a more focused approach by developing a guide RNA library targeting fewer than 200 genes. Their selected targets were all genes previously identified and linked to T-cell fitness, including modulators of transcription, metabolism, cytokine signaling, and exhaustion.

Briefly, the team modified T cells by transducing them with a tumor-specific CAR and silencing the expression of CD3 and target genes from their focused library. Once edited and expanded, the cells were infused into multiple myeloma tumor-bearing mice. Lastly, following the recovery of CAR-T cells from the tumor site or the bone marrow, Mauss and colleagues identified which gene edits led to better CAR-T expansion and persistence over time in vivo.

Interestingly, one of the biggest takeaways from the screen was how context matters.

A prior seminal study by Dr. Alexander Marson’s team at Gladstone-UCSF had identified RASA2 knockout as a top-performing edit in T cells exposed to repeated stimulation in vitro, enhancing their expansion and resistance to exhaustion.⁴ But Dr. Mauss’ team found a different story when they moved to in vivo systems, once RASA2 knockout CAR-T cells were transferred into tumor-bearing mice, the advantage disappeared or even became a disadvantage over time.

This discrepancy highlights the crucial point that screens in cell culture don’t fully capture the complexity of the tumor microenvironment or the pressures CAR-T cells face in living organisms.

Mauss’ team found that among the top-performing gene edits in vivo was CDKN1B, a cell cycle regulator that restricts T cell proliferation. Knocking it out in CAR-T cells led to improved persistence in the bone marrow, greater tumor control, and resistance to exhaustion after repeated antigen exposure. Overall, CDKN1B knockout outperformed previously favored edits like RASA2 in multiple tumor models.

The biggest takeaway from Dr. Mauss’ presentation wasn’t just the gene hits, but her vision for how next-generation CAR-T cell therapies should be discovered in the future. Rather than optimizing CAR-T constructs entirely in the lab, she proposed a “test-drive” model for clinical translation; engineer a batch of CAR-T cells carrying a barcoded pool of gene edits, infuse them into patients, and then use sequencing to track which edits perform best during expansion and tumor clearance.

Such an approach would dramatically accelerate discovery, reduce reliance on imperfect preclinical models, and potentially unlock more effective and durable CAR-T therapies, especially for solid tumors, where progress has been slower.

The lesson is clear: the convergence of the NK and T cell engineering fields on multi-gene editing, clinical "test-drives," and data-driven designs is poised to finally crack solid cancers. CRISPR screens aren’t just accelerating cell therapy; they’re helping to forge two distinct, equally vital arsenals against cancer’s complexity.

While CRISPR screens unlock transformative insights for cell therapy engineering, researchers face significant technical barriers:

Primary Cell Complexity: Low editing efficiency in sensitive immune cells (e.g., NK cells/T cells) and cytotoxicity during transfection.

Functional Relevance Gap: In vitro screens often fail to replicate in vivo tumor microenvironment pressures.

Library Design Risks: Off-target effects, poor coverage, or low-diversity sgRNA pools compromising screen sensitivity.

Scalability: Time-intensive validation and manufacturing delays for custom libraries.

GenScript’s CRISPR Solutions Streamline Discovery:

High-Purity sgRNA Libraries:

• Genome-wide and focused libraries with > 99% construct accuracy.
• Pre-validated guides for enhanced on-target activity (e.g., GeCKO v2, SAM libraries).

Ready-to-Use Formats:

• Lentiviral delivery with high titers for robust cell transduction.

Leverage GenScript’s CRISPR tools to bypass technical bottlenecks and accelerate your path to next-gen cell therapies:
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References

• 1. Peng, L., Sferruzza, G., Yang, L. et al. CAR-T and CAR-NK as cellular cancer immunotherapy for solid tumors. Cell Mol Immunol 21, 1089–1108 (2024). https://doi.org/10.1038/s41423-024-01207-0
• 2. Daher M, Melo Garcia L, Li Y, Rezvani K. CAR-NK cells: the next wave of cellular therapy for cancer. Clin Transl Immunology. 2021 Apr 28;10(4):e1274. doi: 10.1002/cti2.1274
• 3. Acharya S, et al. CD28 Costimulation Augments CAR Signaling in NK Cells via the LCK/CD3ζ/ZAP70 Signaling Axis. Cancer Discov. 2024 Oct 4;14(10):1879-1900. doi: 10.1158/2159-8290.CD-24-0096
• 4. Shanley M, et al. Interleukin-21 engineering enhances NK cell activity against glioblastoma via CEBPD. Cancer Cell. 2024 Aug 12;42(8):1450-1466.e11. doi: 10.1016/j.ccell.2024.07.007
• 5. Hind Rafei, et al. CREM is a regulatory checkpoint of CAR and IL-15 signaling in NK cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1): Abstract nr 6396.Cancer Res (2025) 85 (8_Supplement_1): 6396. https://doi.org/10.1158/1538-7445.AM2025-6396