Figure 4.

T-DNA vector with all the components necessary for Cas9-induced mutagenesis. The 20 bp protospacer sequences of each target site are subcloned or integrated between the sgRNA scaffold and the U3 promoter by ligation of primers into an AarI-digested SK-sgRNA vector. Then, this vector is again re-ligated with a pC1300-Cas9 vector by ligation of BamHI and KnpI-digested enzymes. The whole sgRNA cassette is then delivered into a pC1300-Cas9 vector (contains the Cas9 gene under the control of the 2 × 35S promoter) for plant transformation.