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

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 T-2 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.