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

Enhancing production efficiency, stability, effector function, and other desired features is of prime interest for chimeric antigen receptor engineered T cells (CAR-Ts). Here, we developed a new system for efficient generation of CAR-T with significantly enhanced features by streamlined genome engineering. Leveraging tracrRNA-independent CRISPR/Cpf1 systems with adeno-associated virus (AAV), building a stable CAR-T with homology-directed repair (HDR) knockin and checkpoint knockout (KIKO CAR-T) was achieved at high efficiency in one step. The modularity of the AAV-Cpf1 KIKO system enables flexible and highly efficient generation of double knockin of two different CARs in the same T cell. Compared to Cas9-based methods, the AAV-Cpf1 system generates double knockin CAR-Ts more efficiently. Dual-targeting CD22-specific AAV-Cpf1 KIKO CAR-T cells have potency comparable to Cas9 CAR-Ts in cytokine production and cancer cell killing, while expressing lower levels of exhaustion markers. This versatile system opens new capabilities of T cell immune engineering with simplicity and precision.