Fig. 1
Conventional Cas9-mediated KI and double KO induces high rate of translocations. a Triple gene editing strategy to generate allo-CAR T cells by harnessing CRISPR-Cas to target the TRAC, B2M, and CIITA locus, leading to the depletion of the TCR as well as MHC class I and II molecules, respectively. b Increase of unique translocations with increasing number of introduced double-strand breaks (DSBs) (\(n\cdot2+2\cdot\sum\nolimits_{k=0}^n4\cdot\left(n-1\right)\) with n = number of introduced DSBs). c Visualization of the six unique balanced translocations between the three targeted loci. d Representative flow cytometry histograms show editing outcomes 4 days after co-transfection of Cas9 RNPs targeting the TRAC, B2M, and CIITA genes alone (TRAC-KO (Cas9) + MHC dKO (Cas9)) or in combination with a homology-directed repair template (HDRT) that facilitates the insertion of a CD19 transgene (TRAC-CAR KI (Cas9) + MHC dKO (Cas9)) in comparison to mock electroporated cells. The CAR was stained by using an aFC antibody targeting the IgG1 hinge. e Summary of flow cytometry data of 5 individual healthy donors (n = 5 healthy donors). f Percentage of cells that are triple negative or positive for one, two, or all three analyzed surface markers as determined by applying flow cytometry based on Boolean gating. g Frequencies of cells carrying balanced translocations as determined by ddPCR are shown for all six individual translocations and h as the sum of all translocations detected in mock, TRAC-KO (Cas9) + MHC dKO (Cas9) and TRAC-CAR KI (Cas9) + MHC dKO (Cas9) samples from n = 5 donors. Statistical analysis of flow cytometry and ddPCR data from 5 donors was performed using a one-way ANOVA of matched data with Geisser-Greenhouse correction. Multiple comparisons were performed by comparing the mean of each column with the mean of every other column and corrected by the Turkey test. Asterisks represent different p-values calculated in the respective statistical tests (ns: p ≥ 0.05; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001)