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Nat Methods. 2019 Mar;16(3):247-254. doi: 10.1038/s41592-019-0329-7. Epub 2019 Feb 25.

One-step generation of modular CAR-T cells with AAV-Cpf1.

Dai X1,2,3, Park JJ1,2,3,4, Du Y1,2,3, Kim HR1,2,3, Wang G1,2,3, Errami Y1,2,3, Chen S5,6,7,8,9,10,11.

Author information

1
System Biology Institute, Yale University, West Haven, CT, USA.
2
Department of Genetics, Yale University School of Medicine, New Haven, CT, USA.
3
Center for Cancer Systems Biology, Integrated Science & Technology Center, Yale University, West Haven, CT, USA.
4
Yale MD-PhD Program, Yale University, New Haven, CT, USA.
5
System Biology Institute, Yale University, West Haven, CT, USA. sidi.chen@yale.edu.
6
Department of Genetics, Yale University School of Medicine, New Haven, CT, USA. sidi.chen@yale.edu.
7
Center for Cancer Systems Biology, Integrated Science & Technology Center, Yale University, West Haven, CT, USA. sidi.chen@yale.edu.
8
Yale MD-PhD Program, Yale University, New Haven, CT, USA. sidi.chen@yale.edu.
9
Immunobiology Program, Yale University, New Haven, CT, USA. sidi.chen@yale.edu.
10
Yale Comprehensive Cancer Center, Yale University, New Haven, CT, USA. sidi.chen@yale.edu.
11
Yale Stem Cell Center, Yale University, New Haven, CT, USA. sidi.chen@yale.edu.

Abstract

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.

PMID:
30804551
PMCID:
PMC6519746
[Available on 2019-08-25]
DOI:
10.1038/s41592-019-0329-7

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