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

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.