Fig. 3.

Evaluation of SaCas9 cutting efficiency in HEK293Tcells. The mutation frequency of sgRNAs was evaluated using T7 endonuclease I assay (A), and Sanger sequencing (B) in HEK293T cells transfected with the VVT1-αI, VVT1-αII, and BPK2139, a human expression vector for S.aureus Cas9, was a gift from Keith Joung (Addgene plasmid # 65776) [8]. The expected band sizes of gRNA-gI and gRNA-gII after digestion with T7 enzyme were 177 bp, 181 bp, and 226 bp, 127 bp, respectively. However, no difference was observed before and after digestion by T7 enzyme.