Fig. 8: Efficient engineering of plant architecture through HDR. | Communications Biology

Fig. 8: Efficient engineering of plant architecture through HDR.

From: Fusion of the Cas9 endonuclease and the VirD2 relaxase facilitates homology-directed repair for precise genome engineering in rice

Fig. 8

a Schematic of the desired mutations in the wild-type OsCCD7 allele. Modifications of nucleotides added into the repair templates at the sgRNA binding and PAM sequences to avoid retargeting of the repaired sequence by VirD2-Cas9 are shown. The sgRNA binding sequence is indicated by an arrow and removal of the PAM and insertion of the EcoRI recognition sequence (underlined) is indicated by a line. Nucleotide modifications along with the corresponding amino acids are represented by capital, colored, and bold letters. The respective wild-type amino acids (capital and bold) are also shown. b Representative gels of EcoRI digestion for HDR confirmation. Target (CCD7) flanking 445 bp PCR amplified fragments were subjected to EcoRI digestion. EcoRI digestion confirmed the proper digestion to produce 270 + 175 bp fragments (indicated by arrowheads) and repair in the plants regenerated from calli bombarded with repair templates with mT-RB and Cas9-VirD2 or VirD2-Cas9. Repair templates without mT-NRB with VirD2-Cas9 and Cas9 with mT-RB repair templates were used as control. Lines confirmed with EcoRI digestion are indicated with asterisks. c Photos of plants with engineered modifications. Seed of the progeny plants were grown on soil in the greenhouse and pictures were taken 40 days after germination. d Heritability of the repaired modifications. The target (CCD7) flanking PCR amplified 445-bp fragments from the progeny plants of the selected lines were subjected to EcoRI digestion. EcoRI digestion produced 270 + 170 bp fragments in lines that had inherited the modification. Four out seventeen plants are homozygous for the EcoRI site. e Alignment of the Sanger sequencing reads and representative chromatograms of the EcoRI digestion of confirmed homozygous plants. The repair-specific nucleotide modifications are shown in blue and their exact locations are indicated by arrowheads (red). The representative chromatograms show the exact repair at the targeted locus.

Back to article page