Fig. 4

Eliminating Cas12a target sequence in the HDR template increases KI efficacy. a Design of the dsDNA template for TRAC targeted insertion is shown. The original template containing a mutated Cas9 PAM but an intact Cas12a PAM was modified by mutating the Cas12a PAM on the right homology arm to improve HDR efficiency by preventing cleavage of the repair template. b Representative flow cytometry histograms show KI efficiency using a template with an intact Cas12a PAM and either a Cas9 or Cas12a nuclease to target the TRAC gene. The CAR was stained by using an aFC antibody targeting the IgG1 hinge. c Knock-in efficiency quantified by flow cytometry using the intact or mutated template with a Cas9 nuclease (intact: n = 3, mutated: n = 5 individual donors; unpaired t-test). Empty shapes were performed with the old HDRT, filled shapes were performed with PAM-mutated HDRT. d Representative flow cytometry histograms show KI efficiency using a template with the mutated Cas12a PAM and either a Cas9 or Cas12a nuclease to target the TRAC gene. e Knock-in efficiency using the intact or mutated template with a Cas12a nuclease (intact: n = 3, mutated: n = 5 individual donors; unpaired t-test). f Fold change of KI efficiency with a donor template containing the mutated Cas12a PAM (n = 5) in comparison to the mean (n = 3) of the KI efficiency with the intact template (paired t-test). g Fold change increase of KI efficiency by using Cas12a instead of Cas9 as a nuclease with the HDRT with mutated PAM (n = 5). 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)