File S1 - Targeted Genome Editing of Sweet Orange Using Cas9/sgRNA

The Supporting Information contains Tables S1–S2 and Figures S1–S5. Figure S1, The sequence of the CaMV 35S promoter-CsPDS-targeting sgRNA-NosT of the Cas9/sgRNA construct. The CaMV 35S promoter is shown in blue. The guide sequence is shown in red. The sgRNA scaffold is shown in purple. The Nos terminator is shown in green. The transcription start site is marked by an arrow. Figure S2, Two replicates of the experiment presented in Figure 2 (b). Restriction-enzyme-digestion-suppressed PCR 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. 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 MfeI, which indicates the expected disruption of the MfeI site within the CsPDS gene. M, 1 kb DNA ladder. Figure S3, Two replicates of the experiment presented in Figure 2(d). Measurement of the mutation rate of the CsPDS gene induced by Cas9/sgRNA. After PCR amplification of the targeted PDS region, the products were subjected to MfeI digestion. After separation on an agarose gel, the intensities of the bands were quantified using AlphaImager EP. The mutation rate was calculated by dividing the intensity of the uncut band by the intensity of all the bands in the lane. M, 1 kb DNA ladder. Figure S4, The representative indel chromatograms of the CsPDS12 mutations inducted by Cas9/sgRNA. The target sequence within the CsPDS gene is highlighted by an orange rectangle. Figure S5, Analysis of potential off-target sequences of the CsPDS-targeting Cas9/sgRNA by MfeI-suppressed PCR. (a) Eight potential off-target sequences were amplified by PCR when non-digested genomic DNA was used as the template. (b) When MfeI-digested genomic DNA was used, no PCR products or alleviated PCR products were observed. (c) After selective PCR amplification of mutagenized CsPDS genes, only the PCR product from the CsPDS gene showed resistance to MfeI digestion. These results indicated that the MfeI restriction sites in the 8 potential off-target sequences were not disrupted by Cas9/sgRNA cleavage and NHEJ repair. Table S1, Primers used for Cas9/sgRNA-mediated genome engineering in sweet orange. Table S2, Forty-six potential off-target sequences of the CsPDS gene in the sweet orange genome. (PDF)