Progress and Future Prospects of CRISPR Gene Editing Technology in CAR-T Cell Immunotherapy
Main Article Content
Keywords
CRISPR/Cas9, gene editing, CAR-T cells, immunotherapy, universal CAR-T
Abstract
Since its inception, chimeric antigen receptor T-cell (CAR-T) therapy has achieved remarkable efficacy in relapsed and refractory hematologic malignancies. However, issues such as low response rates in solid tumors, lengthy and costly autologous product preparation, and insufficient long-term safety data have consistently limited its broader clinical application. The maturation of CRISPR/Cas9 gene editing technology has provided new tools to address these bottlenecks—by simultaneously modifying multiple targets in the T-cell genome, researchers can construct off-the-shelf allogeneic CAR-T products, knock out immune checkpoints to enhance antitumor activity, integrate safety switches to control toxicity, and even expand indications to autoimmune diseases. This article focuses on the technical approaches for optimizing CAR-T cells through gene editing. It reviews the current state of research on three strategies—the construction of universal products, enhancement of therapeutic efficacy, and improvement of safety—and, drawing on the latest clinical data from the fields of hematologic malignancies, solid tumors, and autoimmune diseases, discusses unresolved issues such as off-target effects, T-cell exhaustion, and manufacturing processes. The article also provides an outlook on cutting-edge directions, including in vivo CAR-T cells and iPSC-derived cells.
References
- [1]June, C. H., O'Connor, R. S., Kawalekar, O. U., Ghassemi, S., & Milone, M. C. (2018). CAR T cell immunotherapy for human cancer. Science, 359(6382), 1361–1365. https://doi.org/10.1126/science.aar6711
- [2]Neelapu, S. S., Locke, F. L., Bartlett, N. L., Lekakis, L. J., Miklos, D. B., Jacobson, C. A., Braunschweig, I., Oluwole, O. O., Siddiqi, T., Lin, Y., Timmerman, J. M., Stiff, P. J., Friedberg, J. W., Flinn, I. W., Goy, A., Hill, B. T., Smith, M. R., Deol, A., Farooq, U., . . . Go, W. Y. (2017). Axicabtagene ciloleucel CAR T-cell therapy in refractory large B-cell lymphoma. The New England Journal of Medicine, 377(26), 2531–2544. https://doi.org/10.1056/NEJMoa1707447
- [3]Gill, S., & June, C. H. (2015). Going viral: Chimeric antigen receptor T-cell therapy for hematological malignancies. Immunological Reviews, 263(1), 68–89. https://doi.org/10.1111/imr.12243
- [4]Jackson, H. J., Rafiq, S., & Brentjens, R. J. (2016). Driving CAR T-cells forward. Nature Reviews Clinical Oncology, 13(6), 370–383. https://doi.org/10.1038/nrclinonc.2016.36
- [5]Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 337(6096), 816–821. https://doi.org/10.1126/science.1225829
- [6]Doudna, J. A., & Charpentier, E. (2014). The new frontier of genome engineering with CRISPR-Cas9. Science, 346(6213), Article 1258096. https://doi.org/10.1126/science.1258096
- [7]Torikai, H., Reik, A., Liu, P.-Q., Zhou, Y., Zhang, L., Maiti, S., Huls, H., Miller, J. C., Kebriaei, P., Rabinovitch, B., Lee, D. A., Champlin, R. E., Bonini, C., Naldini, L., Rebar, E. J., Gregory, P. D., Holmes, M. C., & Cooper, L. J. N. (2012). A foundation for universal T-cell based immunotherapy: T cells engineered to express a CD19-specific chimeric-antigen-receptor and eliminate expression of endogenous TCR. Blood, 119(24), 5697–5705. https://doi.org/10.1182/blood-2012-01-405365
- [8]Rupp, L. J., Schumann, K., Roybal, K. T., Gate, R. E., Ye, C. J., Lim, W. A., & Marson, A. (2017). CRISPR/Cas9-mediated PD-1 disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells. Scientific Reports, 7(1), Article 737. https://doi.org/10.1038/s41598-017-00462-8
- [9]Shifrut, E., Carnevale, J., Tobin, V., Roth, T. L., Woo, J. M., Bui, C. T., Li, P. J., Diolaiti, M. E., Ashworth, A., & Marson, A. (2018). Genome-wide CRISPR screens in primary human T cells reveal key regulators of immune function. Cell, 175(7), 1958–1971.e15. https://doi.org/10.1016/j.cell.2018.10.024
- [10]Anzalone, A. V., Koblan, L. W., & Liu, D. R. (2020). Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors. Nature Biotechnology, 38(7), 824–844. https://doi.org/10.1038/s41587-020-0561-9
- [11]Frangoul, H., Altshuler, D., Cappellini, M. D., Chen, Y.-S., Domm, J., Eustace, B. K., Foell, J., de la Fuente, J., Grupp, S., Handgretinger, R., Ho, T. W., Kattamis, A., Kernytsky, A., Lekstrom-Himes, J., Li, A. M., Locatelli, F., Mapara, M. Y., de Montalembert, M., Rondelli, D., . . . Corbacioglu, S. (2021). CRISPR-Cas9 gene editing for sickle cell disease and β-thalassemia. The New England Journal of Medicine, 384(3), 252–260. https://doi.org/10.1056/NEJMoa2031054
- [12]Gasiunas, G., Barrangou, R., Horvath, P., & Siksnys, V. (2012). Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proceedings of the National Academy of Sciences, 109(39), E2579–E2586. https://doi.org/10.1073/pnas.1208507109
- [13]Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X., Jiang, W., Marraffini, L. A., & Zhang, F. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science, 339(6121), 819–823. https://doi.org/10.1126/science.1231143
- [14]Anders, C., Niewoehner, O., Duerst, A., & Jinek, M. (2014). Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature, 513(7519), 569–573. https://doi.org/10.1038/nature13579
- [15]Sander, J. D., & Joung, J. K. (2014). CRISPR-Cas systems for editing, regulating and targeting genomes. Nature Biotechnology, 32(4), 347–355. https://doi.org/10.1038/nbt.2842
- [16]Cullot, G., Boutin, J., Toutain, J., Prat, F., Pennamen, P., Rooryck, C., Teichmann, M., Rousseau, E., Lamrissi-Garcia, I., Guyonnet-Duperat, V., Bibeyran, A., Lalanne, M., Prouzet-Mauléon, V., Turcq, B., Ged, C., Blouin, J.-M., Richard, E., Dabernat, S., Moreau-Gaudry, F., & Bedel, A. (2019). CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations. Nature Communications, 10(1), Article 1136. https://doi.org/10.1038/s41467-019-09006-2
- [17]Rees, H. A., & Liu, D. R. (2018). Base editing: Precision chemistry on the genome and transcriptome of living cells. Nature Reviews Genetics, 19(12), 770–788. https://doi.org/10.1038/s41576-018-0059-1
- [18]Engel, N. W., Steinfeld, I., Ryan, D., Anupindi, K., Kim, S., Wellhausen, N., Chen, L., Wilkins, K., Baker, D. J., Rommel, P. C., Jarocha, D., Gohil, M., Zhang, Q., Milone, M. C., Fraietta, J. A., Davis, M., Young, R. M., & June, C. H. (2025). Quadruple adenine base-edited allogeneic CAR T cells outperform CRISPR/Cas9 nuclease-engineered T cells. Proceedings of the National Academy of Sciences, 122(20), Article e2427216122. https://doi.org/10.1073/pnas.2427216122
- [19]Anzalone, A. V., Randolph, P. B., Davis, J. R., Sousa, A. A., Koblan, L. W., Levy, J. M., Chen, P. J., Wilson, C., Newby, G. A., Raguram, A., & Liu, D. R. (2019). Search-and-replace genome editing without double-strand breaks or donor DNA. Nature, 576(7785), 149–157. https://doi.org/10.1038/s41586-019-1711-4
- [20]Gao, Y., Ma, Y., Yu, K., Liu, Y., Gu, B., Tang, H., Yan, W., Yang, S., Su, J., Wang, X., Ma, X., Wang, X., Wang, F., Li, Q., Liu, M., & Wang, H. (2026). Efficient and precise programmable DNA knock-in without double-strand breaks. Nature, 657(8130), 284–294. https://doi.org/10.1038/s41586-026-10819-7
- [21]Goudy, L., Ha, A., Borah, A. A., Umhoefer, J. M., Chow, L., Tran, C., Winters, A., Talbot, A., Hernandez, R., Li, Z., Subramanya, S., Arab, A., Kale, N., Lee, J. H. J., Muldoon, J. J., Liu, C., Schmidt, R., Santangelo, P., Carnevale, J., . . . Gilbert, L. A. (2026). Integrated epigenetic and genetic programming of primary human T cells. Nature Biotechnology, 44(9), 1470–1481. https://doi.org/10.1038/s41587-025-02856-w
- [22]Benjamin, R., Graham, C., Yallop, D., et al. (2016). Generation of an allogeneic universal CAR T cell product using TALEN gene editing. Cancer Immunology Research, 4(11), 926–935.
- [23]MacLeod, R. A., Rawlings, D. J., & Scharenberg, A. M. (2017). Engineering the genome of human T cells for therapy. Molecular Therapy, 25(7), 1592–1603.
- [24]Poirot, L., Philip, B., Schiffer-Mannioui, C., Le Clerre, D., Chion-Sotinel, I., Derniame, S., Potrel, P., Bas, C., Lemaire, L., Galetto, R., Lebuhotel, C., Eyquem, J., Cheung, G. W.-K., Duclert, A., Gouble, A., Arnould, S., Peggs, K., Pule, M., Scharenberg, A. M., & Smith, J. (2015). Multiplex genome-edited T-cell manufacturing platform for “off-the-shelf” adoptive T-cell immunotherapies. Cancer Research, 75(18), 3853–3864. https://doi.org/10.1158/0008-5472.can-14-3321
- [25]Figliola, M. J., Binder-Scholl, G., Plesa, G., et al. (2015). Tetracycline-inducible caspase9 suicide gene for clinical grade B-cell maturation antigen-specific chimeric antigen receptor T cells. Human Gene Therapy, 26(11), 735–744.
- [26]CRISPR Therapeutics (2025, December 22). CRISPR Therapeutics provides broad update on zugocaptagene geleucel (Zugo-cel; formerly CTX112™) in autoimmune diseases and hematologic malignancies [Press release]. https://ir.crisprtx.com/news-releases/news-release-details/crispr-therapeutics-provides-broad-update-zugocaptagene-geleucel
- [27]Hu, Y., Zhou, Y., Zhang, M., Ge, W., Li, Y., Yang, L., Wei, G., Han, L., Wang, H., Yu, S., Chen, Y., Wang, Y., He, X., Zhang, X., Gao, M., Yang, J., Li, X., Ren, J., & Huang, H. (2021). CRISPR/Cas9-engineered universal CD19/CD22 dual-targeted CAR-T cell therapy for relapsed/refractory B-cell acute lymphoblastic leukemia. Clinical Cancer Research, 27(10), 2764–2772. https://doi.org/10.1158/1078-0432.ccr-20-3863
- [28]Zhao, Z., Chen, Y., Francisco, N. M., Zhang, Y., & Wu, M. (2018). The application of CAR-T cell therapy in hematological malignancies: Advantages and challenges. Acta Pharmaceutica Sinica B, 8(4), 539–551. https://doi.org/10.1016/j.apsb.2018.03.001
- [29]Lu, Y., Xue, J., Deng, T., Zhou, X., Yu, K., Deng, L., Huang, M., Yi, X., Liang, M., Wang, Y., Shen, H., Tong, R., Wang, W., Li, L., Song, J., Li, J., Su, X., Ding, Z., Gong, Y., . . . Mok, T. (2020). Safety and feasibility of CRISPR-edited T cells in patients with refractory non-small-cell lung cancer. Nature Medicine, 26(5), 732–740. https://doi.org/10.1038/s41591-020-0840-5
- [30]Shen, X., Fu, W., Wei, Y., et al. (2021). TIGIT-Fc promotes antitumor immunity. Cancer Immunology Research, 9(9), 1088–1097. https://doi.org/10.1158/2326-6066.CIR-20-0986
- [31]Liu, Q., et al. (2026). In vivo genome-wide CRISPR screens of human T cells in solid tumours. Nature, 658(8135), 496–507. https://doi.org/10.1038/s41586-026-10906-9
- [32]Chen, J., López-Moyado, I. F., Seo, H., Lio, C.-W. J., Hempleman, L. J., Sekiya, T., Yoshimura, A., Scott-Browne, J. P., & Rao, A. (2019). NR4A transcription factors limit CAR T cell function in solid tumours. Nature, 567(7749), 530–534. https://doi.org/10.1038/s41586-019-0985-x
- [33]Jones, B. S., Lamb, L. S., Goldman, F., & Di Stasi, A. (2014). Improving the safety of cell therapy products by suicide gene transfer. Frontiers in Pharmacology, 5, Article 254. https://doi.org/10.3389/fphar.2014.00254
- [34]Choe, J. H., Watchmaker, P. B., Simic, M. S., Gilbert, R. D., Li, A. W., Krasnow, N. A., Downey, K. M., Yu, W., Carrera, D. A., Celli, A., Cho, J., Briones, J. D., Duecker, J. M., Goretsky, Y. E., Dannenfelser, R., Cardarelli, L., Troyanskaya, O., Sidhu, S. S., Roybal, K. T., . . . Lim, W. A. (2021). SynNotch-CAR T cells overcome challenges of specificity, heterogeneity, and persistence in treating glioblastoma. Science Translational Medicine, 13(591), Article eabe7378. https://doi.org/10.1126/scitranslmed.abe7378
- [35]Gardner, R., Wu, D., Cherian, S., Fang, M., Hanafi, L.-A., Finney, O., Smithers, H., Jensen, M. C., Riddell, S. R., Maloney, D. G., & Turtle, C. J. (2016). Acquisition of a CD19-negative myeloid phenotype allows immune escape of MLL-rearranged B-ALL from CD19 CAR-T-cell therapy. Blood, 127(20), 2406–2410. https://doi.org/10.1182/blood-2015-08-665547
- [36]Kadirkamanathan, R., Georgiadis, C., Kloos, A., Joshi, A., Etuk, A., Preece, R., Gough, O., Schambach, A., Sauer, M., Heuser, M., & Qasim, W. (2025). Base edited “universal” donor CAR T-cell strategies for acute myeloid leukaemia. Leukemia, 39(12), 2978–2987. https://doi.org/10.1038/s41375-025-02720-5
- [37]Labanieh, L., Majzner, R. G., & Mackall, C. L. (2018). Programming CAR-T cells to kill cancer. Nature Biomedical Engineering, 2(6), 377–391. https://doi.org/10.1038/s41551-018-0235-9
- [38]Chmielewski, M., Kopecky, C., Hombach, A. A., & Abken, H. (2011). IL-12 release by engineered T cells expressing chimeric antigen receptors can effectively muster an antigen-independent macrophage response on tumor cells that have shut down tumor antigen expression. Cancer Research, 71(17), 5697–5706. https://doi.org/10.1158/0008-5472.can-11-0103
- [39]Chinnasamy, D., Yu, Z., Kerkar, S. P., Zhang, L., Morgan, R. A., Restifo, N. P., & Rosenberg, S. A. (2012). Local delivery of interleukin-12 using T cells targeting VEGF receptor-2 eradicates multiple vascularized tumors in mice. Clinical Cancer Research, 18(6), 1672–1683. https://doi.org/10.1158/1078-0432.ccr-11-3050
- [40]Srour, S. A., et al. (2026). Allogeneic CD70-targeted chimeric antigen receptor T-cell therapy for advanced renal cell carcinoma: Results from the phase I TRAVERSE trial. Journal of Clinical Oncology. Advance online publication.
- [41]Mackensen, A., Müller, F., Mougiakakos, D., et al. (2022). Anti-CD19 CAR T cell therapy for refractory systemic lupus erythematosus. Nature Medicine, 28(10), 2124–2132. https://doi.org/10.1038/s41591-022-02017-5
- [42]Wang, X., Zhang, Y., Lin, X., & Xu, H. (2025). Allogeneic CD19-targeting T cells for treatment-refractory systemic lupus erythematosus: A phase 1 trial. Nature Medicine. Advance online publication.
- [43]Müller, F., et al. (2026). CD19 CAR-T cells for treatment-refractory autoimmune diseases: The phase 1/2 CASTLE basket trial. Nature Medicine, 32(3), 1142–1151. https://doi.org/10.1038/s41591-025-04185-6
- [44]Becilli, M., Metzler, M., Bracaglia, C., Nicolai, R., Krickau, T., Del Bufalo, F., Rosignoli, C., Marasco, E., Naumann-Bartsch, N., Messia, V., Merli, P., Pagliara, D., Algeri, M., Secinaro, A., Cefalo, M. G., Diomedi Camassei, F., Bertaina, V., Sinibaldi, M., Insalaco, A., . . . Locatelli, F. (2026). Anti-CD19 CAR T cells for pediatric patients with treatment-refractory autoimmune diseases. Nature Medicine, 32(3), 1105–1117. https://doi.org/10.1038/s41591-025-04191-8
- [45]Chen, K. Y., Kibayashi, T., Giguelay, A., et al. (2025). Genome-wide CRISPR screen in human T cells reveals regulators of FOXP3. Nature, 642(8066), 191–200. https://doi.org/10.1038/s41586-025-08795-5
- [46]Zheng, Z., Liu, Y., Wang, G., Li, X., Peng, S., Jia, Z., Yue, X., Fu, Z., Zhao, X., Han, M., Yang, Z., Tang, C., Gao, J., Kong, Z., Chen, C., Zhao, K., Li, T., Jiang, X., & Jing, W. (2026). In vivo CAR-Tfh cell reprogramming restores tolerance in a mouse model of autoimmune hepatitis. Cell Stem Cell, 33(4), 589–604.e5. https://doi.org/10.1016/j.stem.2026.02.006
- [47]Agarwal, S., Hanauer, J. D. S., Frank, A. M., Riechert, V., Thalheimer, F. B., & Buchholz, C. J. (2020). In vivo generation of CAR T cells selectively in human CD4+ lymphocytes. Molecular Therapy, 28(8), 1783–1794. https://doi.org/10.1016/j.ymthe.2020.05.005
- [48]An, N., Wang, D., Zhang, P., et al. (2026). In vivo generation of anti-BCMA CAR-T cells in relapsed or refractory multiple myeloma: A phase 1 study. Nature Medicine, 32(4), 1257–1266. https://doi.org/10.1038/s41591-026-04244-6
- [49]Pfeiffer, A., Thalheimer, F. B., Hartmann, S., et al. (2018). In vivo generation of human CD19-CAR T cells results in B-cell depletion and signs of cytokine release syndrome. EMBO Molecular Medicine, 10(11), e9158. https://doi.org/10.15252/emmm.201809158
- [50]Themeli, M., Kloss, C. C., Ciriello, G., Fedorov, V. D., Perna, F., Gonen, M., & Sadelain, M. (2013). Generation of tumor-targeted human T lymphocytes from induced pluripotent stem cells for cancer therapy. Nature Biotechnology, 31(10), 928–933. https://doi.org/10.1038/nbt.2678
- [51]Jebran, A. F., Seidler, T., Tiburcy, M., et al. (2025). Engineered heart muscle allografts for heart repair in primates and humans. Nature, 639(8054), 503–511. https://doi.org/10.1038/s41586-024-08463-0
- [52]Montel-Hagen, A., et al. (2019). Organoid-induced differentiation of conventional T cells from human pluripotent stem cells. Cell Stem Cell, 24(3), 376–389. https://doi.org/10.1016/j.stem.2019.01.007
- [53]Kuscu, C., Arslan, S., Singh, R., Thorpe, J., & Adli, M. (2014). Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease. Nature Biotechnology, 32(7), 677–683. https://doi.org/10.1038/nbt.2916
- [54]Grünewald, J., Zhou, R., Garcia, S. P., Iyer, S., Lareau, C. A., Aryee, M. J., & Joung, J. K. (2019). Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors. Nature, 569(7756), 433–437. https://doi.org/10.1038/s41586-019-1161-z
- [55]Zhang, X.-H., Tee, L. Y., Wang, X.-G., Huang, Q.-S., & Yang, S.-H. (2015). Off-target effects in CRISPR/Cas9-mediated genome engineering. Molecular Therapy—Nucleic Acids, 4(11), Article e264. https://doi.org/10.1038/mtna.2015.37
- [56]Charlesworth, C. T., Deshpande, P. S., Dever, D. P., Camarena, J., Lemgart, V. T., Cromer, M. K., Vakulskas, C. A., Collingwood, M. A., Zhang, L., Bode, N. M., Behlke, M. A., Dejene, B., Cieniewicz, B., Romano, R., Lesch, B. J., Gomez-Ospina, N., Mantri, S., Pavel-Dinu, M., Weinberg, K. I., & Porteus, M. H. (2019). Identification of preexisting adaptive immunity to Cas9 proteins in humans. Nature Medicine, 25(2), 249–254. https://doi.org/10.1038/s41591-018-0326-x
- [57]Wherry, E. J., & Kurachi, M. (2015). Molecular and cellular insights into T cell exhaustion. Nature Reviews Immunology, 15(8), 486–499. https://doi.org/10.1038/nri3862
- [58]Martinez, G. J., Pereira, R. M., Äijö, T., Kim, E. Y., Marangoni, F., Pipkin, M. E., Togher, S., Heissmeyer, V., Zhang, Y. C., Crotty, S., Lamperti, E. D., Ansel, K. M., Mempel, T. R., Lähdesmäki, H., Hogan, P. G., & Rao, A. (2015). The transcription factor NFAT promotes exhaustion of activated CD8+ T cells. Immunity, 42(2), 265–278. https://doi.org/10.1016/j.immuni.2015.01.006
- [59]Khan, O., Giles, J. R., McDonald, S., Manne, S., Ngiow, S. F., Patel, K. P., Werner, M. T., Huang, A. C., Alexander, K. A., Wu, J. E., Attanasio, J., Yan, P., George, S. M., Bengsch, B., Staupe, R. P., Donahue, G., Xu, W., Amaravadi, R. K., Xu, X., . . . Wherry, E. J. (2019). TOX transcriptionally and epigenetically programs CD8+ T cell exhaustion. Nature, 571(7764), 211–218. https://doi.org/10.1038/s41586-019-1325-x
- [60]Levine, B. L., Miskin, J., Wonnacott, K., & Keir, C. (2017). Global manufacturing of CAR T cell therapy. Molecular Therapy—Methods & Clinical Development, 4, 92–101. https://doi.org/10.1016/j.omtm.2016.12.006
- [61]Shah, N. N., & Fry, T. J. (2019). Mechanisms of resistance to CAR T cell therapy. Nature Reviews Clinical Oncology, 16(6), 372–385. https://doi.org/10.1038/s41571-019-0184-6
- [62]Qasim, W., et al. (2025). Long-term outcomes of genome-edited universal CAR19 T cells for relapsed/refractory B-cell acute lymphoblastic leukaemia at a single paediatric centre. Blood Advances, 9(18), 4750. https://doi.org/10.1182/bloodadvances.2025016366
