Virus-free CRISPR knockin of a chimeric antigen receptor into KLRC1 generates potent GD2-specific natural killer cells

Keerthana Shankar¹, Isabelle Zingler-Hoslet², Diana M Tabima³, Seth Zima⁴, Lei Shi⁵, Kirstan Gimse³, Matthew H Forsberg⁵, Varun Katta⁶, Sage Z Davis⁷, Daniel Maldonado⁸, Brittany E Russell², Muhammed Murtaza⁹, Shengdar Q Tsai⁶, Jose M Ayuso⁴, Christian M Capitini¹⁰, Krishanu Saha¹¹

Affiliations

¹ Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53715, USA; Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI 53715, USA.
² Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53715, USA.
³ Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI 53715, USA.
⁴ Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53715, USA; Department of Dermatology, University of Wisconsin-Madison, Madison, WI 53715, USA; Carbone Cancer Center, University of Wisconsin-Madison, Madison, WI 53715, USA.
⁵ Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, WI 53715, USA; Carbone Cancer Center, University of Wisconsin-Madison, Madison, WI 53715, USA.
⁶ Department of Hematology, St Jude Children's Research Hospital, Memphis, TN 38105, USA.
⁷ Center for Human Genomics and Precision Medicine, University of Wisconsin-Madison, Madison, WI 53705, USA.
⁸ Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI 53715, USA; Department of Pathobiological Sciences, University of Wisconsin-Madison, Madison, WI 53706, USA.
⁹ Center for Human Genomics and Precision Medicine, University of Wisconsin-Madison, Madison, WI 53705, USA; Department of Surgery, University of Wisconsin-Madison, Madison, WI 53792, USA.
¹⁰ Department of Dermatology, University of Wisconsin-Madison, Madison, WI 53715, USA; Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, WI 53715, USA.
¹¹ Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53715, USA; Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI 53715, USA; Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, WI 53715, USA. Electronic address: ksaha@wisc.edu.

PMID: 39815622
PMCID: PMC11897758 (available on 2026-03-05)
DOI: 10.1016/j.ymthe.2025.01.024

Virus-free CRISPR knockin of a chimeric antigen receptor into KLRC1 generates potent GD2-specific natural killer cells

Keerthana Shankar et al. Mol Ther. 2025.

. 2025 Mar 5;33(3):1014-1030.

doi: 10.1016/j.ymthe.2025.01.024. Epub 2025 Jan 14.

Authors

Affiliations

¹ Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53715, USA; Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI 53715, USA.
² Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53715, USA.
³ Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI 53715, USA.
⁴ Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53715, USA; Department of Dermatology, University of Wisconsin-Madison, Madison, WI 53715, USA; Carbone Cancer Center, University of Wisconsin-Madison, Madison, WI 53715, USA.
⁵ Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, WI 53715, USA; Carbone Cancer Center, University of Wisconsin-Madison, Madison, WI 53715, USA.
⁶ Department of Hematology, St Jude Children's Research Hospital, Memphis, TN 38105, USA.
⁷ Center for Human Genomics and Precision Medicine, University of Wisconsin-Madison, Madison, WI 53705, USA.
⁸ Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI 53715, USA; Department of Pathobiological Sciences, University of Wisconsin-Madison, Madison, WI 53706, USA.
⁹ Center for Human Genomics and Precision Medicine, University of Wisconsin-Madison, Madison, WI 53705, USA; Department of Surgery, University of Wisconsin-Madison, Madison, WI 53792, USA.
¹⁰ Department of Dermatology, University of Wisconsin-Madison, Madison, WI 53715, USA; Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, WI 53715, USA.
¹¹ Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53715, USA; Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI 53715, USA; Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, WI 53715, USA. Electronic address: ksaha@wisc.edu.

PMID: 39815622
PMCID: PMC11897758 (available on 2026-03-05)
DOI: 10.1016/j.ymthe.2025.01.024

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.

Keywords: CAR; CRISPR-Cas9; GD2; KLRC1; NK cells; NKG2A; non-viral.

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Conflict of interest statement

Declaration of interests K.Saha receives honoraria for advisory board membership for Andson Biotech, Bharat Biotech, and Notch Therapeutics. C.M.C. receives honoraria for advisory board membership for Bayer, Elephas, Nektar Therapeutics, Novartis, and WiCell Research Institute. S.Q.T. receives honoraria and equity from scientific advisory board membership for Prime Medicine and Ensoma. S.Q.T. is one of the co-inventors in a patent describing CHANGE-seq. M.M. consults for the Translational Genomics Research Institute (TGen), a not-for-profit research institution. K.Shankar and K.Saha are inventors on a patent application to be filed with the Wisconsin Alumni Research Foundation (WARF) for the technology in this study.

Update of

Virus-free CRISPR knock-in of a chimeric antigen receptor into KLRC1 generates potent GD2-specific natural killer cells.
Shankar K, Zingler-Hoslet I, Shi L, Katta V, Russell BE, Tsai SQ, Capitini CM, Saha K. Shankar K, et al. bioRxiv [Preprint]. 2024 Feb 14:2024.02.14.580371. doi: 10.1101/2024.02.14.580371. bioRxiv. 2024. Update in: Mol Ther. 2025 Mar 05;33(3):1014-1030. doi: 10.1016/j.ymthe.2025.01.024. PMID: 38405747 Free PMC article. Updated. Preprint.

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Virus-free CRISPR knockin of a chimeric antigen receptor into KLRC1 generates potent GD2-specific natural killer cells

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Virus-free CRISPR knockin of a chimeric antigen receptor into KLRC1 generates potent GD2-specific natural killer cells

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