CRISPR/Cas9-targeted mutagenesis of F3′H, DFR and LDOX, genes related to anthocyanin biosynthesis in black rice (Oryza sativa L.)

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Altering a trait by CRISPR-Cas9-targeted mutagenesis offers great advantages in identifying gene function and crop improvement. In the present study, three genes (OsF3′H, OsDFR and OsLDOX) in the anthocyanin biosynthesis pathway were successfully edited on the Heugseonchal or Sinmyungheugchal variety using the CRISPR/Cas9 system. As a result, the ratio of the edited plants in the transformed early generation was 56.7%. These edited mutant lines were observed with the changes of seed color and anthocyanin content. All mutations were stably inherited to the T2 progeny. In addition, we could select edited homozygous mutant lines lacking the T-DNA already in the first offspring generation. Also the insertion of vector backbone sequences in f3′h-9, dfr-4 and ldox-16 lines was not detected in the whole genome resequencing. These results demonstrated that the CRISPR/Cas9 system can induce clearly gene-specific mutations with a high efficiency in rice and null plants selected from these mutants cannot be distinguished from non-GMO plants even under strict GMO regulation.