Figure 4.

Effect of PAM directions on NHBEJ repair frequency

(A) Schematic outline of the experimental design. The gRNA 1 and 2 were pairs of gRNAs with opposite PAM directions but generating DSBs at the same position. Four regions in DMD intron 44 were selected. (B) PCR-based genotyping results in HEK293T cells targeted with SpCas9 and 24 pairs of gRNAs. PCR was done on a pool of transfected cells with triplicate transfections. An asterisk (∗) indicates DNA bands that were gel-purified and analyzed by Sanger sequencing and ICE. (C) Schematic presentation of the three PAM combinations. (D) Quantification and comparison of NHBEJ efficiency with different PAM orientations. There was no statistical difference between the three groups (ANOVA).