Journal
NATURE METHODS
Volume 16, Issue 3, Pages 247-+Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/s41592-019-0329-7
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Funding
- Yale SBI/Genetics Startup Fund
- Damon Runyon Dale Frey Award [DFS-13-15]
- Melanoma Research Alliance [412806, 16-003524]
- St-Baldrick's Foundation [426685]
- Breast Cancer Alliance
- Cancer Research Institute (CLIP)
- AACR [499395, 17-20-01-CHEN]
- Mary Kay Foundation [017-81]
- V Foundation [V2017-022]
- Ludwig Family Foundation
- US Department of Defense [W81XWH-17-1-0235]
- Sontag Foundation
- Chenevert Foundation
- NIH/NCI [1DP2CA238295-01, 1R01CA231112-01, 1U54CA209992-8697, 5P50CA196530-A10805, 4P50CA121974-A08306]
- CRI Irvington postdoctoral fellowship
- RJ Anderson postdoctoral fellowship
- Yale MSTP training grant from the NIH [T32GM007205]
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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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