Genome Editing Targeting Flavanone 3-Hydroxylase Knock Out Gene Using CRISPR/CAS9 (pRGEB32-F3H) in Rice Plant Transformation

Abstract

Sakuranetin is a flavonoid compound predominantly produced by rice plants in response to biotic and abiotic stress. This compound can be induced through stress triggers such as UV radiation, jasmonic acid accumulation, and pathogen infection. Its biosynthesis originates from the precursor naringenin, catalyzed by the OsNOMT gene. However, naringenin can also be converted into dihydrokaempferol by the Flavanone 3-Hydroxylase (F3H) enzyme, affecting sakuranetin production. The conjugation of naringenin by F3H reduces the accumulation of sakuranetin, a phytoalexin crucial for plant stress resistance. Enhancing sakuranetin levels can be achieved by knocking out or silencing genes like F3H through CRISPR/Cas9 genome editing, a transformative genetic engineering method inducing targeted gene mutations. This research focuses on designing sgRNA for the F3H gene to optimize sakuranetin production in rice. The methodology involves selecting F3H sgRNA targets, constructing target gene mutation plasmids, transforming plasmids into Agrobacterium, and subsequently infecting rice explants. The study yielded an F3H sgRNA sequence with 55% efficiency for insertion into the pRGEB32 plasmid. This plasmid was successfully transformed into Agrobacterium, verified by rifampicin and kanamycin antibiotic selection and PCR confirmation. Infected rice explants from the Koshihikari variety displayed positive responses, evidenced by the formation of planlets, signifying the success of the transformation process.