Targeted mutation of GA20ox-2 gene using CRISPR/Cas9 system generated semi-dwarf phenotype in rice

Recently, the engineered CRISPR/Cas9 system has been applied to rapidly and efficiently modify the targeted gene(s) in a wide variety of plants. Recent studies of successful targeted mutagenesis using the CRISPR/Cas9 system with a single gRNA expression in rice plants have been reported. GA20ox-2 is a gene encoding an oxidase enzyme involved in the biosynthesis of gibberellin and linked to sd1 locus. A previous study revealed that mutation of this gene resulted in shorter stature of rice plant due to defects in the gibberellin’s signalling pathway. Here, we studied targeted mutation of OsGA20ox-2 gene in rice using the CRISPR/Cas9 system with the expression of two gRNAs. In this study, we introduced a single plasmid vector of CRISPR/Cas9 system harboring dual gRNAs to modify OsGA20ox-2 gene in a rice model cv. Kitaake via Agrobacterium -mediated transformation. Targeted mutagenesis of OsGA20ox-2 gene using CRISPR/Cas9 generated nine mutated rice lines with a mutation frequency of 90%. Most mutated lines (50%) had mutations in both OsGA20ox-2 gRNA. They resulted in homo-diallelic mutation type with 44 bp deletion, while three lines were heterozygous, one line was homo-diallelic with 2 bp insertion, and one line had no mutation. The K15 mutated rice line was identified as a homozygous two-nucleotide insertion and had the semi-dwarf phenotype, demonstrating that OsGA20ox-2 gene had been disrupted.