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

Abstract

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.

Genome‐wide association and genomic prediction for yield and component traits of Miscanthus sacchariflorus

“…Internode elongation is an important determinant of plant height and overall biomass yield for bioenergy grasses (Salas Fernandez et al, 2009). In rice, loss of function mutants for the GA synthesis genes GA20 and G1D1 produced a dwarf phenotype (Qin et al, 2013; Santoso et al, 2020; Sasaki et al, 2002). Conversely, overexpression of GID1 in transgenic rice resulted in elevated levels of GA and increased internode elongation (Ueguchi‐Tanaka et al, 2005).…”

Enabling Trade in Gene-Edited Produce in Asia and Australasia: The Developing Regulatory Landscape and Future Perspectives

“…In Indonesia, the earliest published reports on R&D on GEd in agriculture were in 2020 on the rice GA20ox-2 gene (cvs Kitaake and Mentong) encoding a gibberellin biosynthetic enzyme [ 24 , 25 , 26 ] and the OsCKX2 gene in the rice cv Mentik Wangi [ 27 ]. Several research institutes and universities are working on the GEd of different crops.…”

Gibberellic acid sensitive dwarf encodes an ARPC2 subunit that mediates gibberellic acid biosynthesis, effects to grain yield in rice

“…Different expression patterns of GA20ox contribute to phenotypic variation in Arabidopsis ( Rieu et al., 2008 ), with GA20ox overexpression increasing the production of bioactive GAs, which influence plant growth ( Huang et al., 1998 ). In rice, meanwhile, functionally deficient mutants of GA20ox show a typical dwarf phenotype ( Barboza et al., 2013 ; Santoso et al., 2020 ).…”