In vitro SpCas9 cleavage assay

In brief, the plasmids with either the original Psnc1::gfp::snc1 sequence or the SpCas9 PAM site mutated sequence were used as donor DNAs. Each 600 ng were restricted with 10 U NotI in a total volume of 20 µL to generated linearized DNAs. After heat-inactivation (20 min at 80 °C) of NotI, the mixtures were immediately added without further purification to a 30 µL reaction mixture containing 1 µL gRNA and 1 µL Cas9 protein. After 60 min incubation at 37 °C, the reaction was quenched by adding 3 µL 0.5 M EDTA and 7 µL 6× gel loading dye. Samples were incubated for 15 min at 65 °C and analysed by electrophoresis on a 1% agarose gel.