CRISPR (clustered regularly interspaced short palindromic repeats) gene-edited (GEd) crops have demonstrated significant potential to enhance global food security in the face of escalating climate challenges and rapid population growth. Since 2019, for regulatory purposes, the United States (U.S.) and several other countries have recognized transgene-free, genome-edited lines as equivalent to conventionally bred varieties. Notably, the first genome-edited food product, Calyno™ soybean oil, was commercialized in the U.S. and marketed as a non-genetically modified organism (GMO) item. Recently, regulatory frameworks, such as the enactment of the Precision Breeding Law in the United Kingdom, the European Union’s New Genomic Techniques (NGT) legislation, and the repeal of the SECURE Rule in the United States, have further established guidelines permitting the use of genome-edited lines in agriculture similar to with conventionally bred crops, provided that these lines are free of transgenic elements. In Korea, researchers and policymakers are actively engaging in discussions to establish a preliminary review committee for GEd crops to align regulatory practices with international trade standards. Thus, this study aimed to evaluate two gene-edited rice lines for generational stability in terms of molecular characteristics, focusing on edited nucleotide sequences, gene expression, target phenotypes, the presence of transgene elements, and potential off-target effects across multiple generations. Additionally, several technical challenges in nucleotide editing tracing emerged during the evaluation process that warrant further attention. The findings presented in this study are expected to offer valuable insights for shaping the regulatory framework in Korea for CRISPR-based gene-edited crops.
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Resveratrol, a plant phenolic compound, has potential therapeutic benefits due to its antioxidant properties. This is substantiated by previous studies that show that resveratrol derived from rice grains is an effective treatment agent for metabolic syndrome. Here, we characterized the T-DNA sequence, inserted T-DNA structure, copy number, integrity of the transgene locus, resveratrol synthase gene expression and resveratrol contents in the grains of two resveratrol transgenic rice lines, Iksan515 and Iksan526. The T-DNA transformation vector contained two expression cassettes of the resveratrol synthase gene under the control of the ubiquitin promoter and the bar selection marker gene under the control of the CaMV35S promoter. Flanking sequence analysis indicated that the T-DNAs were inserted into intergenic regions of chromosome 4 for Iksan515 and chromosome 12 for Iksan526. Two T-DNAs connected in an inverted repeat structure at a single locus of the rice genome were identified by whole genome sequencing and Southern blot hybridization in both Iksan515 and Iksan526. No novel open reading frames (ORFs) around insertion sites, sequences encoding allergenic or toxic protein, or other unintended effects by T-DNA insertion were found in either case. In addition, resveratrol synthase gene expression in leaves and resveratrol detection in brown rice grains suggested the successful expression of the inserted foreign resveratrol synthase gene in two transgenic rice lines.
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