3.2 Activity assay of rSaCas9
The activity assay of rSaCas9 is based on sequence-specific cleavage (linearization) of plasmid DNA. Therefore, cell-free produced rSaCas9 was tested on ten different target sites within an ~ 1 kb promoter sequence of the CCD8 gene from Z. mays L. Suitable target sites within that sequence were identified using the Cas designer tool (http://www.rgenome.net/cas-designer/). Figure S3 shows the target sites, including the PAM sequence 5’-NNGRRT-3’. Six target sites were derived from the plus strand, and four sites were derived from the minus strand. The corresponding constructs for the in vitrotranscription of the analogous sgRNAs are shown in Figure S4. For the activity assay, the rSaCas9 enzyme was incubated with the individual sgRNAs and the plasmid containing the target sequence. Agarose gel electrophoresis confirmed that the plasmid DNA had been cleaved (linearized) in the presence of each of the ten sgRNAs and rSaCas9, represented by the expected 4 kb band (Figure 3). In contrast, the control samples without rSaCas9 or without the addition of sgRNA showed no signs of cleavage, as indicated by the higher mobility on the agarose gel due to the more compact conformation of the noncleaved plasmid DNA.