The preparation of the proteins and the cleavage assay were performed by following a previous study . In brief, the sequence containing the AAAGAA PAM was first cloned into vector pUC19 (referred as pUC-AGAA). The plasmids with distinct PAM sites were constructed by site specific mutagenesis, using pUC-AGAA as the template (Table ). The plasmids were linearized by KpnI digestion and the products were used as the cleavage substrates. In vitro cleavage reactions were performed in a 10 μL reaction buffer (10 mM Tris-HCl (pH 7.5), 500 mM NaCl, 1.5 mM MgCl2, 1 mM DTT) containing 5 nM linearized plasmids, 250 nM purified St1Cas9 or its variants and 500 nM sgRNA. The reactions were incubated at 37 °C for 40 min and quenched by 25 mM EDTA and 10 μg Proteinase K for incubation at 58 °C for 10 min. Reaction products were analyzed by a 1% agarose gel.