Extended Data Fig. 8: Severe phenotypes resulting from GoSAMT3 CRISPR-Cas9 gene editing in gosamt1 gosamt2 plants.
(A) Schematic diagram of the GoSAMT3 gene. Green arrowheads show sgRNA targets, and blue arrows show primer positions for genotyping experiments. The red arrowhead shows T-DNA position of the gosamt3 mutant described in Supplemental Fig. 7. (B) Schematic diagram of the CRISPR-Cas9 construct. Transcriptional units were assembled into L2 vectors using Golden Gate Modular Cloning. (C) Representative images of 16-day-old WT, gosamt1 gosamt2 mutant plants, and WT and gosamt1 gosamt2 plants after transformation with the GoSAMT3 CRISPR-Cas9 construct. A range of severity of phenotypes was seen, only in the transformed gosamt1 gosamt2 mutants, which were grouped into three severity classes. (D) Quantitation of individual phenotypes and ungerminated seeds from the GoSAMT3 CRISPR-Cas9 transformed gosamt1 gosamt2 seeds. (E) Representative scanning electron microscope images of seedlings in group 3. Bar = 500μm Similar results were observed in three different plants of this group. (F) GoSAMT3 genotyping of WT, gosamt1 gosamt2, gosamt3 and three GoSAMT3 CRISPR-Cas9 transformed WT (#1, #2 and #3) and gosamt1 gosamt2 mutant plants (#5, #6, #7). Deletions indicating partial gene editing were observed in all T1 individuals. The blue asterisk corresponds to WT GoSAMT3 amplicon size using the primers shown in A. Red asterisks correspond to GoSAMT3 amplification products containing deletions. (G) Sanger sequencing of the PCR products from individuals in (F), revealing deletion of regions of the GoSAMT3 gene.
