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Massively parallel knock-in engineering of human T cells

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单位： [1]Department of Genetics, Yale University School of Medicine, New Haven, CT, USA. [2]System Biology Institute, Yale University, West Haven, CT, USA. [3]Center for Cancer Systems Biology, Yale University, West Haven, CT, USA. [4]M.D.-Ph.D. Program, Yale University, West Haven, CT, USA. [5]Molecular Cell Biology, Genetics, and Development Program, Yale University, New Haven, CT, USA. [6]Department of Chemistry, Yale University, New Haven, CT, USA. [7]Institute of Biomolecular Design and Discovery, Yale University, West Haven, CT, USA. [8]Department of Biostatistics, Yale University School of Public Health, New Haven, CT, USA. [9]Immunobiology Program, Yale University, New Haven, CT, USA. [10]Computational Biology and Bioinformatics Program, Yale University, New Haven, CT, USA. [11]Yale Center for Biomedical Data Science, Yale University School of Medicine, New Haven, CT, USA. [12]Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA. [13]Combined Program in the Biological and Biomedical Sciences, Yale University, New Haven, CT, USA. [14]Department of Neurosurgery, Yale University School of Medicine, New Haven, CT, USA. [15]Yale Comprehensive Cancer Center, Yale University School of Medicine, New Haven, CT, USA. [16]Yale Stem Cell Center, Yale University School of Medicine, New Haven, CT, USA. [17]Yale Liver Center, Yale University School of Medicine, New Haven, CT, USA. [18]Yale Center for RNA Science and Medicine, Yale University School of Medicine, New Haven, CT, USA. [19]Present address: Department of Thyroid and Breast Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. [20]Present address: Department of Colorectal and Anal Surgery, The First Affiliated Hospital of Guangxi Medical University, Nanning, China.

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The efficiency of targeted knock-in for cell therapeutic applications is generally low, and the scale is limited. In this study, we developed CLASH, a system that enables high-efficiency, high-throughput knock-in engineering. In CLASH, Cas12a/Cpf1 mRNA combined with pooled adeno-associated viruses mediate simultaneous gene editing and precise transgene knock-in using massively parallel homology-directed repair, thereby producing a pool of stably integrated mutant variants each with targeted gene editing. We applied this technology in primary human T cells and performed time-coursed CLASH experiments in blood cancer and solid tumor models using CD3, CD8 and CD4 T cells, enabling pooled generation and unbiased selection of favorable CAR-T variants. Emerging from CLASH experiments, a unique CRISPR RNA (crRNA) generates an exon3 skip mutant of PRDM1 in CAR-Ts, which leads to increased proliferation, stem-like properties, central memory and longevity in these cells, resulting in higher efficacy in vivo across multiple cancer models, including a solid tumor model. The versatility of CLASH makes it broadly applicable to diverse cellular and therapeutic engineering applications.© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.

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大类 | 1 区

工程技术

小类 | 1 区生物工程与应用微生物

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大类 | 1 区

生物学

小类 | 1 区生物工程与应用微生物

第一作者：

第一作者单位： [1]Department of Genetics, Yale University School of Medicine, New Haven, CT, USA. [2]System Biology Institute, Yale University, West Haven, CT, USA. [3]Center for Cancer Systems Biology, Yale University, West Haven, CT, USA.

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通讯机构： [1]Department of Genetics, Yale University School of Medicine, New Haven, CT, USA. [2]System Biology Institute, Yale University, West Haven, CT, USA. [3]Center for Cancer Systems Biology, Yale University, West Haven, CT, USA. [4]M.D.-Ph.D. Program, Yale University, West Haven, CT, USA. [5]Molecular Cell Biology, Genetics, and Development Program, Yale University, New Haven, CT, USA. [9]Immunobiology Program, Yale University, New Haven, CT, USA. [11]Yale Center for Biomedical Data Science, Yale University School of Medicine, New Haven, CT, USA. [13]Combined Program in the Biological and Biomedical Sciences, Yale University, New Haven, CT, USA. [14]Department of Neurosurgery, Yale University School of Medicine, New Haven, CT, USA. [15]Yale Comprehensive Cancer Center, Yale University School of Medicine, New Haven, CT, USA. [16]Yale Stem Cell Center, Yale University School of Medicine, New Haven, CT, USA. [17]Yale Liver Center, Yale University School of Medicine, New Haven, CT, USA. [18]Yale Center for RNA Science and Medicine, Yale University School of Medicine, New Haven, CT, USA.

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