Extended Data Fig. 2: Viability, editing, and activation metrics with non-viral intron editing and negative selection in human T cells.

a, Timeline of primary human T cell editing using non-viral intron knock-ins, followed by negative selection. T cells are isolated and activated on Day 0, followed by electroporation based non-viral intron editing on Day 2. CD3 Negative selection was performed on Day 8, six days followed editing. b, Total T cell counts on Day 1 and Day 4 post electroporation relative to no electroporation controls following non-viral intron knock-ins by electroporation of a TRAC intron targeting CRISPR-Cas9 RNP along with a DNA Homology Directed Repair Template. Approximately half of the cell loss observed in the intron knock-in condition (“RNP + DNA”) is due to electroporation itself, and half due to DNA toxicity, as observed previously for non-viral exon knock-ins using electroporation in primary human T cells^5,14. n = 3 donors with 3 technical replicates each. c, The percentage of successfully edited intron knock-in cells across timepoints, beginning three days after editing (Day 5 post activation). Across n = 3 donors, editing percentages were stable in culture during post-editing expansion. Error bars represent standard error of the mean. d, At Day 8 post activation, TRAC intron knock-in T cells were negatively selected by binding magnetic beads to the CD3 complex. Compared to the input population prior to selection, post-negative selection intron knock-in T cells did not show any decrease in viability as measured by Tryphan Blue staining and Live/Dead dye flow cytometric staining. The observed increase in viability after selection is likely due to washing steps removing dying cells present in the input culture. e, After either negative selection or no selection, the activation status of TRAC intron knock-in CD19-28z CAR T cells was assessed through in vitro activation by Nalm6 target cells at a 1:4 Effector:Target cell ratio. 24 hours post co-incubation, CD69 and CD25 expression levels in CAR positive (tNGFR + ) and CAR negative (tNGFR-) cells was assayed by flow cytometry. n = 2 unique donors with 3 technical replicates. ns = not significant, Two-sided paired t test.