speedingCARs: accelerating the engineering of CAR T cells by signaling domain shuffling and single-cell sequencing

Due to the low-throughput process of systematically designing and functionally testing chimeric antigen receptors (CARs), only a small set of immune signaling domains have been thoroughly explored, despite their major role in T cell activation, effector function and persistence. SpeedingCARs presents an integrated method for engineering CAR T cells by signaling domain shuffling and functional screening by single-cell sequencing. Leveraging the inherent modularity of natural signaling domains, we generated a diverse library of 180 unique CAR variants, which were genomically integrated into primary human T cells by CRISPR-Cas9. Functional and pooled screening of the CAR T cell library was performed by co-culture with tumor cells, followed by single-cell RNA sequencing (scRNA-seq) and single-cell CAR sequencing (scCAR-seq), thus enabling high-throughput profiling of multi-dimensional cellular responses. A library of 180 unique CAR variants was genomically integrated into primary human T cells by CRISPR-Cas9 genome editing. Single-cell RNA sequencing data of pooled library CAR T cells from 3 different healthy donors was generated following 36h of co-culture with HER2 expressing breast cancer cell line SKBR3. As negative controls TCR negative T cells and CD28-CD3Z unstimulated CAR T cells were also sequenced.