Immune-cell engineering opens new capabilities for fundamental immunology research and immunotherapy. We developed a system for efficient generation of chimeric antigen receptor (CAR)-engineered T cells (CAR-T cells) with considerably enhanced features by streamlined genome engineering. By leveraging trans-activating CRISPR (clustered regularly interspaced short palindromic repeats) RNA (tracrRNA)-independent CRISPR–Cpf1 systems with adeno-associated virus (AAV), we were able to build a stable CAR-T cell with homology-directed-repair knock-in and immune-checkpoint knockout (KIKO CAR-T cell) at high efficiency in one step. The modularity of the AAV–Cpf1 KIKO system enables flexible and highly efficient generation of double knock-in of two different CARs in the same T cell. Compared with Cas9-based methods, the AAV–Cpf1 system generates double-knock-in CAR-T cells more efficiently. CD22-specific AAV–Cpf1 KIKO CAR-T cells have potency comparable to that of Cas9 CAR-T cells in cytokine production and cancer cell killing, while expressing lower levels of exhaustion markers. This versatile system opens new capabilities of T-cell engineering with simplicity and precision.
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Analytic codes used to generate figures that support the findings of this study will be made available by the corresponding author upon reasonable request.
Genome sequencing data are available via SRA/BioProject under accession number PRJNA509600. Plasmids and libraries are being deposited to Addgene. A list of AAV vectors generated and used in this study is provided in Supplementary Table 2. Original and processed data are included in the figures, figure legends, and supplementary materials of this article. Other relevant data and materials that support the findings of this study will be made available by the corresponding author upon reasonable request.
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We thank C. Fuchs and R. Herbst for their assistance and insightful discussions. We thank L. Ye, J. Li, L. Shen, M. Dong, R. Chow, Z. Bai, X. Zhang, and all other members of the Chen laboratory for technical assistance and discussions. We thank various colleagues in the Department of Genetics, Systems Biology Institute, Cancer Systems Biology Center, MCGD Program, Immunobiology Program, BBS Program, Cancer Center, and Stem Cell Center at Yale for assistance and/or discussion. We thank the Center for Genome Analysis, Center for Molecular Discovery, Pathology Tissue Services, Histology Services, High Performance Computing Center, West Campus Analytical Chemistry Core and West Campus Imaging Core, and Keck Biotechnology Resource Laboratory at Yale for technical support. S.C. is supported by the Yale SBI/Genetics Startup Fund, the Damon Runyon Dale Frey Award (grant/award number DFS-13-15), the Melanoma Research Alliance (412806, 16-003524), St-Baldrick’s Foundation (426685), the Breast Cancer Alliance, the Cancer Research Institute (CLIP), AACR (499395, 17-20-01-CHEN), the Mary Kay Foundation (017-81), the V Foundation (V2017-022), the Ludwig Family Foundation, the US Department of Defense (W81XWH-17-1-0235), the Sontag Foundation, the Chenevert Foundation, and the NIH/NCI (1DP2CA238295-01, 1R01CA231112-01, 1U54CA209992-8697, 5P50CA196530-A10805, 4P50CA121974-A08306). G.W. is supported by CRI Irvington and RJ Anderson postdoctoral fellowships. J.J.P. is supported by a Yale MSTP training grant from the NIH (no. T32GM007205).