DNA cleavage assay was carried out based on [3 (link)] with commercially available Cas9 enzyme (NEB). PCR amplified genomic fragments for each sgRNA-1 off-target site (OT#1_F 5’-AGAGGCAAGTAAAGGTCAAGTAGG-3’ , OT#1_R 5’-TCACATTGCAATGATGAGCACTTT-3’ ; OT#2_F 5’-CCAGCTCATGTTGAAAAGACACAT-3’ , OT#2_R 5’-CCCCCACAGATGAAATGAAAAGAC-3’ ; OT#3_F 5’-TACCCAAAAATTGTAAGCCAGCAG-3’ , OT#3_R 5’-AGATCTGATCCGGTTTCAAAGTGA-3’ ) were cloned into pGEM-T easy vector (Promega). The plasmids were pre-linearized with BsaI (NEB) about 2kb from the sgRNA target site. 3nM of the pre-linearized plasmids were incubated for one 1h with 30nM sgRNA-1 and 30nM Cas9 protein (NEB) and supplemented with Cas9 nuclease buffer (NEB) in a 30μl reaction volume at 37°C. Gel electrophoresis was performed on 1.5% agarose gel in 1x TAE buffer (40mM Tris, 20mM acetic acid, 1mM EDTA).
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