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Here, we established a novel method for generating CAR\u2010\u03b3\u03b4 T cells. We utilized the clustered regularly interspaced palindromic repeats (CRISPR)\/CRISPR\u2010associated protein 9 (Cas9) (CRISPR\/Cas9) method to interrupt the\n                    <jats:italic>TCR delta chain constant region<\/jats:italic>\n                    (\n                    <jats:italic>TRDC<\/jats:italic>\n                    ) sequence, followed by the site\u2010directed insertion of the CAR sequence into the\n                    <jats:italic>TRDC<\/jats:italic>\n                    locus via homologous complementation mediated by adeno\u2010associated virus (AAV) gene delivery. We optimized electroporation parameters for Cas9\/ribonucleoproteins (RNP) delivery and infection conditions for CAR\u2010gene carrying AAV in \u03b3\u03b4 T cells. These optimizations facilitated efficient TCR knockout and site\u2010directed CAR insertion, ultimately yielding functional CAR\u2010\u03b3\u03b4 T cells. In\u00a0vitro experiments demonstrated that these newly prepared CAR\u2010\u03b3\u03b4 T cells could stimulate cytokine production, kill tumor cells as well as exhibit robust proliferative potential and memory\u2010like phenotype. These state\u2010of\u2010the\u2010art CAR\u2010\u03b3\u03b4 T cells could reduce tumor burden and extend the survival period of tumor\u2010bearing mice.\n                  <\/jats:p>","DOI":"10.1002\/cam4.71918","type":"journal-article","created":{"date-parts":[[2026,5,18]],"date-time":"2026-05-18T09:11:36Z","timestamp":1779095496000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["A Novel Strategy to Produce\n                    <scp>CAR<\/scp>\n                    \u2010\u03b3\u03b4 T Cells via Site\u2010Directed Gene Integration by a Combination of\n                    <scp>CRISPR<\/scp>\n                    \/Cas9 and\n                    <scp>AAV<\/scp>"],"prefix":"10.1002","volume":"15","author":[{"given":"Ruoyu","family":"Dong","sequence":"first","affiliation":[{"name":"Department of Hematology and Cell Therapy, Sir Run Run Shaw Hospital Zhejiang University School of Medicine  Hangzhou Zhejiang Province China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yixi","family":"Zhang","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, National Medical Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital Zhejiang University School of Medicine  Hangzhou China"},{"name":"Department of Developmental Biology and Cancer, Great Ormond Street Institute of Child Health University College London  London UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guangxun","family":"Yuan","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, National Medical Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital Zhejiang University School of Medicine  Hangzhou China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaoyan","family":"Yue","sequence":"additional","affiliation":[{"name":"Department of Hematology and Cell Therapy, Sir Run Run Shaw Hospital Zhejiang University School of Medicine  Hangzhou Zhejiang Province China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yingying","family":"Ding","sequence":"additional","affiliation":[{"name":"Department of Hematology and Cell Therapy, Sir Run Run Shaw Hospital Zhejiang University School of Medicine  Hangzhou Zhejiang Province China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7292-1819","authenticated-orcid":false,"given":"Xun","family":"Zeng","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, National Medical Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital Zhejiang University School of Medicine  Hangzhou China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Haowen","family":"Xiao","sequence":"additional","affiliation":[{"name":"Department of Hematology and Cell Therapy, Sir Run Run Shaw Hospital Zhejiang University School of Medicine  Hangzhou Zhejiang Province China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,5,18]]},"reference":[{"key":"e_1_2_10_2_1","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2023.1045024"},{"key":"e_1_2_10_3_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41571\u2010022\u201000704\u20103"},{"key":"e_1_2_10_4_1","doi-asserted-by":"publisher","DOI":"10.1200\/JCO.2018.77.8084"},{"key":"e_1_2_10_5_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2020.08.022"},{"key":"e_1_2_10_6_1","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2022.889051"},{"key":"e_1_2_10_7_1","doi-asserted-by":"publisher","DOI":"10.1038\/mt.2012.267"},{"key":"e_1_2_10_8_1","doi-asserted-by":"publisher","DOI":"10.1002\/eji.1830271111"},{"key":"e_1_2_10_9_1","doi-asserted-by":"publisher","DOI":"10.1172\/JCI35798"},{"key":"e_1_2_10_10_1","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMoa2405541"},{"key":"e_1_2_10_11_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkv1121"},{"key":"e_1_2_10_12_1","doi-asserted-by":"publisher","DOI":"10.1038\/nature21405"},{"key":"e_1_2_10_13_1","doi-asserted-by":"publisher","DOI":"10.3791\/3182"},{"key":"e_1_2_10_14_1","doi-asserted-by":"publisher","DOI":"10.3892\/mmr.2019.9831"},{"key":"e_1_2_10_15_1","doi-asserted-by":"publisher","DOI":"10.1158\/2326\u20106066.CIR\u201018\u20100466"},{"key":"e_1_2_10_16_1","doi-asserted-by":"publisher","DOI":"10.1007\/s13238\u2010019\u20100643\u2010y"},{"key":"e_1_2_10_17_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41598\u2010017\u201000462\u20108"},{"key":"e_1_2_10_18_1","doi-asserted-by":"publisher","DOI":"10.1182\/blood\u20102017\u201001\u2010761320"},{"key":"e_1_2_10_19_1","doi-asserted-by":"publisher","DOI":"10.1084\/jem.20171626"},{"key":"e_1_2_10_20_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467\u2010023\u201044176\u20100"},{"key":"e_1_2_10_21_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.celrep.2014.02.040"},{"key":"e_1_2_10_22_1","doi-asserted-by":"publisher","DOI":"10.1038\/nprot.2017.143"},{"key":"e_1_2_10_23_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ymthe.2017.12.001"},{"key":"e_1_2_10_24_1","first-page":"79","article-title":"MUC1\u2010Tn\u2010Targeting 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