Generation of rice mutants using the CRISPR/Cas9 method
For the generation of perox4 and osmt1a/b knockout lines,
guide RNAs (gRNAs) (Supplemental Table 4) were designed to target exons
of the targeted genes (Fig. S3). The synthesized oligo of gRNAs was
annealed to form the oligo adaptors, which then was joined to the BGK03
CRISPR/Cas9 vector (Biogle Biotechnology, Hangzhou, China). The
CRISPR/Cas9 plasmids were introduced into Agrobacterium
tumefaciens EHA105 and then into rice throughAgrobacterium- mediated transformation as described previously.
The individual T1 plants were genotyped by sequencing the DNA products
of PCR with primers (Supplemental table 4).