Abstract
Editing chimeric antigen receptor (CAR) T cells by using CRISPR–Cas9 has become a routine strategy to improve their antitumor function or safety profile. Xenograft tumor models in immunodeficient mice are often used to evaluate the function of CRISPR-edited human CAR T cells. These models, however, lack functional immune systems and thus fail to recapitulate barriers such as the immunosuppressive tumor microenvironment (TME) that CAR T cells will encounter in patients. Thus, genetically modifying mouse CAR T cells for use in immune-intact models is an attractive approach to explore the impact of a given gene deletion on CAR T cells within a natural TME. Here, we describe a protocol to perform CRISPR–Cas9 editing in primary mouse T cells, thereby enabling studies of gene-edited CAR T within the TME and in the presence of a functional immune system. This protocol is integrated into a standard mouse CAR T manufacturing workflow, a process that typically spans ~5–6 days. The first stage of this protocol involves isolating mouse T cells, electroporating them with a ribonucleoprotein complex and activating them by using magnetic bead stimulation. The second stage involves transducing the CAR gene and expanding these cells, and the third stage focuses on validating knockout efficiency and the functionality of gene-edited mouse CAR T cells. This procedure requires a proficiency in aseptic cell culture techniques and a basic understanding of T cell biology. We anticipate that efficient and reliable genetic modification of mouse T cells will have wide-ranging applications for cancer immunotherapies and related fields.
Key points
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This protocol involves isolating mouse T cells and electroporation of Cas9 RNPs, followed by activation with beads and retroviral transduction of a CAR construct.
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It enables rapid gene deletion in mouse CAR T cells that is nontoxic, preserves T cell fitness, maintains adequate growth kinetics for proper quality control before these cells undergo functional testing and allows the impact of a given gene deletion on CAR T cells to be assessed within a natural tumor microenvironment.
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Data availability
Example data that can be generated with this protocol are available from the corresponding author on reasonable request. Other materials such as vector sequences can be requested but may be subject to a Materials Transfer Agreement. Source data are provided with this paper.
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Acknowledgements
We acknowledge I. Maillard and his laboratory for the gift of CD45.1+ BALB/c mice. Furthermore, this research was supported by the NSF Graduate Research Fellowship Program (NSF-GRFP), DGE 1845298 (to P.G.); NIH R00CA212302 and R01-37- CA262362-03 (to M.R.); the Laffey McHugh Foundation (to M.R.); the Berman and Maguire Funds for Lymphoma Research at Penn (to M.R.); and NIH/NCI R01CA197916 (to G.L.B), R01CA245323 (to G.L.B) and F31CA271692 (to J.P.). Figure 1 was created by using BioRender.com.
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P.G. and M.R. conceptualized the project, interpreted and discussed the results and drafted the manuscript. P.G., R.R., S.N., A.C., S.L., L.P., V.H., J.P. and R.P. performed the experiments. V.B. and G.L.B. provided baseline protocols and reagents and interpreted the results. M.R. funded and supervised the project. All authors reviewed and approved the manuscript.
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Competing interests
M.R. reports receiving grants from viTToria Biotherapeutics, AbClon and Oxford Nanoimaging. He also reports nonfinancial support (equipment) from Beckman Coulter, LUMICKS and Curiox Biosystems. In addition, M.R. serves on the Scientific Advisory Boards of viTToria Biotherapeutics, AbClon, Acera and LUMICKS. He receives consulting fees from GLG and Guidepoint and holds CAR T-related patents managed by the University of Pennsylvania, which are partly licensed to Novartis, Kite and viTToria Biotherapeutics. All other authors declare no competing interests.
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Key references
Guruprasad, P. et al. Nat. Immunol. 25, 1020–1032 (2024): https://doi.org/10.1038/s41590-024-01847-4
Patel, R. P. et al. Sci. Immunol. 9, eadn6509 (2024): https://doi.org/10.1126/sciimmunol.adn6509
Extended data
Extended Data Fig. 1 Effect of extrinsic and intrinsic factors affecting CRISPR-edited murine CAR T cell expansion, viability, purity and engraftment.
a, Fold change (FC; left) and cell volume (right) on Day 6 of expanding murine untransduced (UTD) cells that received No EP, Mock EP, or EP with each CD5 gRNA candidate (4 × 106 BALB/c T cells per condition). b, Flow cytometry plots depicting enrichment of CD5-negative fraction following 5 day manufacturing of CD5 KO murine CAR T cells and subsequent bead-based positive selection (Anti-PE MultiSort Kit, Miltenyi Biotec cat no. 130-090-757). c,d, The effects of cytokines on the fold change (c) and viability (d) of murine CAR T cells were evaluated on Day 6 of manufacturing across three electroporation (EP) conditions: No EP, Mock EP, and CD5 KO. T cells were first subjected to one of these EP conditions, with two electroporation replicates performed for each arm, using 10 × 106 cells per replicate. Post-electroporation, T cells from each arm were seeded into wells at 2 × 106 cells per well. Each well was supplemented with media containing one or more of three cytokines — IL-2, IL-7, or IL-15 — each at a concentration of 10 ng/ml. Cytokines were present during transduction and expansion, and their effects on cell fold change and viability were assessed at day 6. e, Fold change of CD5 KO mouse T cell expansion, with portions de-beaded and quantified on each days 4, 5, and 6 of the protocol (4 × 106 BALB/c T cells per condition). f, Effect of mouse age on CD5-negative fraction, CAR+ fraction, viability, and fold change of cell number expansion of CD5− mouse CART19 at day 5 of manufacturing. Here, T cells from spleens of BALB/c mice of various ages (10, 29, and 42 weeks old) were isolated, and electroporation was performed (4 × 106 BALB/c murine T cells per condition, n = 2 mice of each age, and 1 electroporation per mouse). g, Effect of cryopreservation on viability and expansion of CD5− mouse CART19 at day 5 of manufacturing (4 × 106 BALB/c murine T cells per condition, n = 2 electroporation replicates). Here, cryopreserved BALB/c T cells were thawed in IL-2 + IL-7 (10 ng/ml each) for 2 h prior to EP. Both frozen and fresh BALB/c T cells came from 10-week-old BALB/c mice. h, Fold change and viability in murine CAR T cells, comparing electroporation of RNP before activation (day 0) and after activation/expansion (day 5). Following de-beading (and EP for post-activation EP arm), cells in each condition were resuspended at 1 × 106 cells per ml in fresh media containing IL-2 and IL-7 (10 ng/ml each). Fold change and viability were re-assessed at day 7. i,j, In vivo experiment to measure tumor infiltration of CRISPR-edited murine CART19 based on lymphodepletion (LD) administration of cytoxan/cyclophosphamide (CTX), using the A20 BALB/c-derived large B cell lymphoma model. These experiments were carried out as described in ref. 11. Briefly, donor CD5-deficient CART19 cells were generated using CD45.1+ BALB/c mice, and infused into CD45.2+ (wild-type) A20-bearing BALB/c hosts. 2 × 106 A20 were implanted per mouse to establish subcutaneous tumors, and subsequently infused 18 days post-implantation with 1 × 106 4-1BBζ CAR19+ T cells. Tumors were resected on 5 days post-infusion and T cell infiltration was measured via flow cytometry. i, Representative flow plots of resected A20 tumors of mice who received no treatment, CART19 without CTX LD, and CART19 with CTX LD (n = 2 mice per condition). Cells here are gated previously on live T cells (ViaKrome−, CD3+). j, Quantification of CD45.1+ (adoptively transferred) and CD45.2+ (host) tumor infiltrating lymphocytes (TIL) in each condition. All experiments here used 10 µg Cas9 + 5µg CD5 sgRNA (gRNA1, except for a) to construct the RNP per EP; mock EP used 5 µg Cas9 only. Error bars represent mean +/− s.d.
Extended Data Fig. 2 Comparison of mock EP conditions in the generation and function of human CAR T cells.
In brief, CD4 and CD8 T cells from a healthy human donor were mixed in a 1:1 ratio and received No EP, EP with 10 µg Cas9, or EP with 5 µg scramble gRNA + 10 µg Cas9. 5 × 106 total T cells were used per EP condition. Electroporated T cells were allowed to recover for 24 h at 37 °C prior to activation with magnetic anti-human CD3/CD28 beads (2:1 bead to T cell ratio). Half of cells from each electroporation condition were then transduced with human 4-1BBζ CAR19 viral vector. After 6 days, stimulation beads were removed, and cell counts were measured every 2 days until the cells reached a resting state, at which point they were cryopreserved. a, Ex vivo expansion kinetics of UTD and CART19 conditions under each EP configuration. b, T cell memory subset phenotyping at the end of expansion. Subsets here are defined based on cell fractions expressing CCR7 and/or CD45RA (see ref. 11). c,d, Mean in vitro cytotoxicity (luminescence-based) of CART19 cell arms at 24, 48, and 72 h of coculture against Karpass 422 (effector: target = 0.25, n = 3 technical replicates, c) and Nalm6 (effector: target = 0.125, n = 3 technical replicates, d). Here, 50,000 target cells were used per replicate. Significance was determined by one-way ANOVA with post hoc Tukey tests (c,d) at the 72 h timepoint between CART19 arms. ns, non-significant; UTD, untransduced; NHL, non-Hodgkin lymphoma; B-ALL, B cell acute lymphoblastic leukemia; TEMRA, terminally differentiated effector memory T cells.
Extended Data Fig. 3 General gating strategy.
a, Gating for CD5-deficient mouse T cells 5 days post-electroporation with 5 µg CD5 sgRNA + 10 µg Cas9 and subsequent activation. The following order is used for all flow cytometry experiments: side scatter (SSC-A) vs. forward scatter (FSC-A) to gate T cells, FSC-A vs. FSC-H to gate single cells, FSC-A vs. ViaKrome 808 to gate for live cells. All data were captured on the Beckman Coulter CytoFLEX LX Flow Cytometer, and analyses were performed using FlowJo version 10.8.1 (FlowJo, LLC).
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Full western blot for Fig. 3d
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Guruprasad, P., Ramasubramanian, R., Nason, S. et al. Manufacturing of CRISPR-edited primary mouse CAR T cells for cancer immunotherapy. Nat Protoc 20, 3629–3654 (2025). https://doi.org/10.1038/s41596-025-01208-x
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DOI: https://doi.org/10.1038/s41596-025-01208-x
