All six sgRNA produce Cas9-driven cleavage in vitro . 200 ng of the PCR product of the LuloYLW gene exon 3 were incubated with each individual sgRNA (3.2 μM) in the absence (lanes 1–6) or presence of 3.8 μM Cas9 protein (lanes 7–12). DNA bands size corresponds to the expected size according to each cleavage site (sgRNA1 = 218 bp, 154 bp; sgRNA2 = 230 bp, 142 bp; sgRNA3 = 294 bp, 78 bp; sgRNA4 = 279 bp, 93; sgRNA5 = 158 bp, 214 bp and sgRNA6 = 148 bp, 224 bp). As negative controls, 200 ng of PCR product alone (lane 13) or in combination with Cas9 protein (lane 14) were included. All samples were run on 2.2% agarose gels and visualized under UV light.