Fig. 6: Schematic overview of speedingCARs: an integrated approach for the rapid engineering of CARs. | Nature Communications

Fig. 6: Schematic overview of speedingCARs: an integrated approach for the rapid engineering of CARs.

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

Fig. 6

a First, an initial CAR architecture is chosen based on functionality and encoded in a DNA plasmid. Next, one or more modular domains of the CAR are swapped for alternative natural immune intracellular signaling domain in a semi-random combinatorial fashion, resulting in a plasmid library of CAR variants with diverse combinations. b The plasmid library of CAR variants is genomically integrated at the TRAC locus of primary human T cells via CRISPR-Cas9 genome editing. This ensures that each cell expresses a single CAR variant, and simultaneously deletes the endogenous TCR. A pooled library of CAR T cells is co-cultured in the presence of tumor cells expressing cognate antigens. c The functional screening of the pooled CAR T cell library is performed by scRNA-seq and scCAR-seq, revealing the transcriptional phenotype. This dataset is used to select promising variants for in-depth characterization with functional assays.

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