Fig. 7: Elimination of chromosomal translocations by Cas9TX in CAR T cells.

a Schematics for the manufacturing of CAR-T cells by SpCas9 or Cas9TX RNPs. Human T cells obtained from human cord blood were activated for 3 days by anti-CD3/CD28, infected by a lentivirus carrying anti-CD19 scFv, and transfected with SpCas9/Cas9TX RNPs. CAR-T killing assays were performed at 3-days post-transfection, and translocations among TRAC, TRBC, and PDCD1 were analyzed by PEM-seq at 3-, 7-, and 14-days post-transfection. See methods for more details. b Percentages of T cells infected by the lentivirus carrying CAR-CFP detected by FACS. Paired two-tailed t-test, mean ± SD from three biological replicates; n.s, no significance. c Editing efficiency at the TRAC, TRBC, and PDCD1 genes at 3-days post-transfection detected by PEM-seq. Paired two-tailed t-test, mean ± SD from three biological replicates; n.s, no significance. d Representative flow cytometry results indicating TCR expression levels at 3-days post-transfection. NC is the negative control without RNP editing. e, f Circos plots indicating translocations among TRAC, TRBC, PDCD1, and TRAC off-targets by SpCas9 (e) or Cas9TX (f) at 3 days after RNP transfection detected by PEM-seq. Mean values for three biological replicates are indicated. Red asterisks indicate the identified TRAC off-target site, depicted as described in the legend to Fig. 1c. g–i The ratios of SpCas9- or Cas9TX-induced translocations of TRAC-PDCD1 (g), TRAC-TRBC (h), and TRAC general translocations (i) cloned from TRAC by PEM-seq at 3-, 7-, and 14-days post-transfection. Mean values for three biological replicates are indicated. Mean ± SD, paired two-tailed t-test, *p < 0.05, **p < 0.01. j The killing capacities of SpCas9- and Cas9TX-editing CAR-T cells were evaluated by CD19⁺ K562 killing assay. T indicates target cells and E indicates effector cells. Mean ± SD from three biological replicates, two-tailed t-test, n.s means no significance.