Method for producing rice plants with increased carotenoid content and environmental stress tolerance using gene editing, and rice plants produced by said method

Rice with Enhanced Carotenoid Content and Oxidative Stress Tolerance via OsOr Gene Editing

This technology involves genome editing by introducing a guide RNA targeting the OsOr gene (SEQ ID NO: 2) and a Cas endonuclease into rice cells, either as a vector or a ribonucleoprotein complex, followed by plant regeneration. The resulting rice and seeds exhibit higher carotenoid levels and greater resistance to oxidative stress compared to wild-type rice.

Carotenoids are photosynthetic accessory pigments, provitamin A sources, and antioxidants that protect cells from reactive oxygen species, yet rice endosperm naturally produces almost none. Previous methods, such as introducing maize PSY and bacterial CrtI (like Golden Rice 2) or overexpressing the Or gene, relied on transgenic modification, which requires costly and time-consuming GMO safety assessments. Furthermore, overexpressing biosynthetic genes, as seen in tomatoes, can lead to growth abnormalities like dwarfism.

This technology uses CRISPR/Cas9 to edit the OsOr gene—which encodes a DnaJ-family chaperone protein that regulates plastid development—to increase carotenoid accumulation and enhance tolerance to environmental stresses like oxidation and salinity without using foreign biosynthetic genes. The induced mutations are indistinguishable from natural variations, allowing them to be treated as non-GMO breeding materials. This approach enables the development of provitamin A-fortified rice, salt-tolerant varieties for reclaimed land, and antioxidant-rich rice for processing, securing both nutritional enhancement and cultivation stability through a single gene-editing event.

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Key Features:

• A recombinant vector comprising DNA encoding a guide RNA specific to the OsOr target sequence of SEQ ID NO: 2 and a nucleic acid sequence encoding an endonuclease
• A genome-editing composition containing a ribonucleoprotein complex of an OsOr-specific guide RNA and an endonuclease protein to increase carotenoid content and oxidative stress tolerance
• A step of introducing a guide RNA specific to the SEQ ID NO: 2 target and an endonuclease protein into rice plant cells to edit the OsOr genome
• A rice plant and its seeds obtained by regenerating the edited rice plant cells, characterized by increased carotenoid content and oxidative stress tolerance

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This invention was developed with support from the Rural Development Administration for the development and efficiency improvement of HDR gene-editing technology in rice.

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