Fig. 1: Combining modular domain shuffling and genome editing to express a library of CAR variants in primary human T cells. | Nature Communications

Fig. 1: Combining modular domain shuffling and genome editing to express a library of CAR variants in primary human T cells.

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

Fig. 1

a Schematic representation of CAR architecture for the shuffling of signaling domains. The CAR variant library is derived from an initial second-generation CAR featuring the intracellular signaling domains of CD28 and CD3ζ; the scFv (4D5) is based on the variable domains of the clinical antibody trastuzumab (specificity to the antigen HER2). The entire CD28 signaling domain and a segment of the CD3ζ domain are exchanged with signaling domains from two pools: Domain A and B, which possess either zero or one ITAM, respectively. A truncated CD3ζ (tCD3Z) possessing two ITAMs is retained. b Combinatorial shuffling of Domain A and Domain B intracellular signaling domains yields a library of 180 possible CAR variants. c Schematic representation of the cloning strategy for domain shuffling. A backbone vector was designed to encode a CAR chassis composed of the following conserved elements: the CD8ɑ secretion peptide, the scFv 4D5, two Strep tags separated by G4S linkers, the CD28 hinge and transmembrane domains and a partial region of CD3ζ. A cloning cassette between the transmembrane domain and CD3ζ domain has outward-facing recognition sequences of the Type IIS restriction enzyme AarI. A restriction digest yields unique overhangs that are compatible with the ligation of one domain from pool A and one domain from pool B, in that order (5’ to 3’). The construct is completed by a polyadenylation signal and flanked by homology arms for the targeted genomic integration of the transgene in the human TRAC locus. d Long-read deep sequencing was performed following cloning and assembly of the signaling domain shuffling library. Circos plot shows that 179/180 possible combinations are present in balanced proportions (Source data are provided as a Source Data file.). e Schematic of the strategy for targeted genomic integration of a CAR library into the TRAC locus of primary human T cells by CRISPR-Cas9 HDR. f, g Flow cytometry sequential gating of human primary T cells after transfection (f) and after selection for CAR expression (g).

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