Fig. 5

Co-delivery of Cas12a for KI and Cas9-derived BE avoids translocations during complex editing. a Experimental setup for the generation of CAR T cells by co-delivery of a Cas9 or Cas12a RNP mediating the TRAC insertion with sgRNAs directing an mRNA encoded ABE to target splice sites of the B2M and CIITA loci (TRAC-CAR KI (Cas9) + MHC dKO (nCas9-BE), TRAC-CAR KI (Cas12a) + MHC dKO (nCas9-BE)). b Representative flow cytometry histograms show editing outcomes of TRAC-CAR KI (Cas9) + MHC dKO (nCas9-BE), TRAC-CAR KI (Cas12a) + MHC dKO (nCas9-BE) and mock electroporated cells. The CAR was stained by using an aFC antibody targeting the IgG1 hinge. c Summary plots for surface expression data from 5 donors. Empty shapes were performed with the old HDRT, filled shapes were performed with PAM-mutated HDRT. 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 translocations detected in mock, TRAC-CAR KI (Cas9) + MHC dKO (nCas9-BE) and TRAC-CAR KI (Cas12a) + 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)