Fig. 1

Comparison of Homology-Directed Recombination versus Homology-independent-targeted-insertion for targeted knock-in of a GD2-CAR into TRAC. a Schematic overview of workflow for experiments in b-d, and nanoplasmid designs for knock-in templates. b-d head-to-head comparison of constructs HDR2c, HITI2c and HITI1c. 5 × 10⁶ cells were electroporated per condition on day 2 post activation using respective constructs (0.75 µg of nanoplasmid per 1 × 10.⁶ cells) and analyzed via flow cytometry on day 10 (b, representative donor; c, pooled frequencies) and counted on the same day to assess GD2-CAR-T cell counts (d) (n = 4 independent donors). e–h HDR inhibitor induced modulation of GD2-CAR-T cell integration via HITI and HDR. e Schematic related to f–h. f Representative histograms of GD2-CAR expression after CRISPR knock-in with HDR2c or HITI1c templates either left untreated or treated with 1 µM of AZD0156 for 18 h post electroporation. g + h GD2-CAR expression (g) and GD2-CAR-T cell counts (h) normalized to untreated CRISPR knock-in samples after 18 h of treatment with indicated concentrations of AZD0156 (n = 3 independent donors). i-j, CRISPR knock-in of non-activated T cells using HITI1c and HDR2c for knock-in of the GD2-CAR. Indicated are knock-in frequencies (i), Viability (j) and GD2-CAR-T cell yield. Cells were counted and analyzed via flow cytometry on day 10 or 14 (n = 4 independent donors). p values were determined by paired two-tailed t tests. Error bars indicate standard deviation (SD)