Fig. 2

Co-delivery of Cas9-derived BE reduces translocations during simultaneous KI. a Experimental setup for the generation of triple-edited CAR T cells by co-delivery of a Cas9 RNP mediating the TRAC insertion with sgRNAs directing an mRNA encoded adenine base editor to target splice sites of the B2M and CIITA loci (TRAC-CAR KI (Cas9) + MHC dKO (nCas9-BE)). b Representative flow cytometry histograms show editing outcomes of TRAC-CAR KI (Cas9) + MHC dKO (Cas9), TRAC-CAR KI (Cas9) + MHC dKO (nCas9-BE) and mock electroporated cells. c Summary plots for surface expression data from 5 donors. The CAR was stained by using an aFC antibody targeting the IgG1 hinge. d 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 Boolean gating. e Frequencies of cells carrying balanced translocations as determined by ddPCR are shown for all six individual translocations and f as the sum of all translocation detected in mock, TRAC-CAR KI (Cas9) + MHC dKO (Cas9) and TRAC-CAR KI (Cas9) + MHC dKO (nCas9-BE) 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)