Fig. 5

CsCMV-mediated genome editing using the CRISPR/Cas9 system in cassava. (a) Schematic representation of the pCsCMV2-gMePDS1 and -gMePDS2 vectors. The protospacer region sequences (marked in red) and the gRNA scaffold sequences (underlined) were cloned downstream of SGP4 into the pCsCMV2-NC vector. (b) Representative photobleaching phenotypes in Cas9-overexpressing (Cas9-OE) transgenic cassava lines agroinfiltrated with CsCMV2-gMePDS1 or -gMePDS2 at 35 dpi. The CsCMV2-GFP-infected plants were used as the controls. (c) The T7 endonuclease 1 (T7E1) mismatch detection assay of CsCMV2-gMePDS1 or -gMePDS2-induced genome editing. Targeted MePDS gene mutagenesis was detected in the photobleached leaves of three representative Cas9-OE transgenic cassava lines agroinfiltrated with CsCMV2-gMePDS1 or -gMePDS2. Arrows indicate the T7EI cleavage products. The indel rate (%) was calculated using the Image J software. (d) Sanger sequencing of wild-type (WT) and mutant versions (M1-M9) of the MePDS gene from photobleached cassava leaf area via CsCMV2-gMePDS1 or -gMePDS2-induced genome editing. The target/mutated sequences are highlighted in light red. The protospacer-associated motif (PAM) is underlined in blue. The number of different nucleotide deletions in the MePDS target sites is indicated on the right