Figure 2. Targeted genome engineering in sweet orange using the Cas9/sgRNA system.

(a) Scheme of the binary vectors 1380-Cas9 and 1380-Cas9:sgRNA. A Flag tag and a nuclear localization signal (NLS) were fused to the Cas9 N-terminus and C-terminus, respectively. Cas9 catalyzes the cleavage of the sgRNA-targeting sequence immediately upstream of the PAM. Here, Cas9/sgRNA was employed to target the CsPDS gene (red). The MfeI restriction site and the protospacer adjacent motif (PAM) are underlined. (b) Selective PCR amplification of mutagenized CsPDS genes was used to detect the Cas9/sgRNA-induced mutation in planta. PCR amplification was conducted using the primers CsPDS-5-P1 and CsPDS-3-P2, which flank the target site within the CsPDS gene (Table S1 in File S1). Lanes 1-3, the template genomic DNA was digested with MfeI. Lane 4, nondigested genomic DNA was used as a template. The PCR product in lane 1 resulted from Cas9/sgRNA-induced disruption of the MfeI site and was therefore cloned into the PCR-BluntII-TOPO vector (Life Technologies) for sequencing. (c) Targeted mutations induced by Cas9/sgRNA in the CsPDS gene in sweet orange. Sequences of mutant variants of the CsPDS gene obtained from the clones constructed using the PCR product from lane 1 in Fig. 2b were aligned with the wild type sequence (top). The sgRNA-targeted CsPDS sequence is shown in red, and the mutations are shown in purple. (d) Measurement of the mutation rate of the CsPDS gene induced by Cas9/sgRNA. Genomic DNA was extracted from three samples (co-expression of Cas9 and sgRNA; expression of Cas9 alone; no expression of Cas9 and sgRNA), and subjected to PCR amplification using the primers CsPDS-5-P1 and CsPDS-3-P2. The PCR products were digested with MfeI and analyzed by DNA gel electrophoresis (Lane 1, co-expression of Cas9 and sgRNA; Lane 2, expression of Cas9 alone; Lane 3, no expression of Cas9 and sgRNA). The mutation rate was calculated by dividing the intensity of the uncut band by the intensity of all the bands in each lane.