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Updated: May 5, 2026
Generation of Human Chimeric Antigen Receptor Regulatory T Cells
Published on: January 3, 2025
Virus-free CRISPR knockin of a chimeric antigen receptor into KLRC1 generates potent GD2-specific natural killer
Keerthana Shankar1, Isabelle Zingler-Hoslet2, Diana M Tabima3
1Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53715, USA; Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI 53715, USA.
Abstract:
Natural killer (NK) cells are an appealing off-the-shelf, allogeneic cellular therapy due to their cytotoxic profile. However, their activity against solid tumors remains suboptimal in part due to the upregulation of NK-inhibitory ligands, such as HLA-E, within the tumor microenvironment. Here, we utilize CRISPR-Cas9 to disrupt the KLRC1 gene (encoding the HLA-E-binding NKG2A receptor) and perform non-viral insertion of a GD2-targeting chimeric antigen receptor (CAR) within NK cells isolated from human peripheral blood. Genome editing with CRISPR-Cas9 ribonucleoprotein complexes yields efficient genomic disruption of the KLRC1 gene with 98% knockout efficiency and specific knockin of the GD2 CAR transgene as high as 23%, with minimal off-target activity as shown by CHANGE-seq, in-out PCR, amplicon sequencing, and long-read whole-genome sequencing. KLRC1-GD2 CAR NK cells display high viability and proliferation, as well as precise cellular targeting and potency against GD2+ human tumor cells. Notably, KLRC1-GD2 CAR NK cells overcome HLA-E-based inhibition in vitro against HLA-E-expressing, GD2+ melanoma cells. Using a single-step, virus-free genome editing workflow, this study demonstrates the feasibility of precisely disrupting inhibitory signaling within NK cells via CRISPR-Cas9 while expressing a CAR to generate potent allogeneic cell therapies against HLA-E+ solid tumors.
Insights
This study engineered natural killer (NK) cells to target solid tumors by disrupting inhibitory signals and adding a chimeric antigen receptor (CAR). These enhanced NK cells show potent anti-tumor activity, overcoming tumor defenses.
Area of Science:
- Immunology
- Cell Therapy
- Genome Editing
Background:
- Natural killer (NK) cells are promising for allogeneic cell therapy but struggle against solid tumors.
- Tumor microenvironments often upregulate inhibitory ligands like HLA-E, dampening NK cell activity.
Purpose of the Study:
- To engineer NK cells to overcome tumor-induced inhibition and enhance anti-tumor efficacy.
- To develop a virus-free genome editing strategy for NK cell therapy.
Main Methods:
- CRISPR-Cas9 was used to knock out the KLRC1 gene (encoding NKG2A) and insert a GD2-targeting chimeric antigen receptor (CAR) into human NK cells.
- Genome editing efficiency and specificity were validated using multiple sequencing and PCR techniques.
Main Results:
- Achieved 98% KLRC1 gene knockout and up to 23% GD2 CAR knockin with minimal off-target effects.
- Engineered KLRC1-GD2 CAR NK cells demonstrated high viability, proliferation, and potent cytotoxicity against GD2+ tumor cells.
- These cells effectively overcame HLA-E-mediated inhibition in vitro.
Conclusions:
- A single-step, virus-free genome editing method enables precise disruption of NK cell inhibitory signaling and CAR insertion.
- This approach generates potent allogeneic NK cell therapies capable of targeting solid tumors expressing HLA-E and GD2.
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