Precise gene insertion using homology-directed repair (HDR) in Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated gene editing has emerged as a powerful tool for plant genome modification, offering unprecedented control over genetic alterations. This study successfully applied this technology to TBR225, a major rice variety in Vietnam, aiming to overexpress the OsNRAMP7 gene by inserting a 35S constitutive promoter. Our research addressed the need for efficient methods to enhance specific gene expression in crop plants, with a focus on improving this economically important rice variety. We designed and implemented a single vector system comprising gRNA and Cas9 expression constructs, along with a donor DNA repair template containing the 35S promoter. The system was delivered via Agrobacterium-mediated transformation to TBR225 rice. Our results demonstrated the successful insertion of the 35S promoter upstream of the OsNRAMP7 open reading frame in TBR225, leading to significantly increased OsNRAMP7 expression in edited lines. Notably, we obtained homozygous transgene-free OsNRAMP7-overexpressing TBR225 lines in the T1 generation, confirming stable transmission of the desired modification in this Vietnamese rice variety. These results validate the effectiveness of CRISPR/Cas9-mediated HDR for precise genetic modifications in TBR225 rice. This successful research on Vietnam's major rice variety offers a valuable approach for crop improvement and functional genomics studies, potentially accelerating the development of rice varieties with enhanced traits such as stress tolerance or nutrient use efficiency.
Key words: CRISPR/Cas9, Gene overexpression, Homology-directed repair (HDR), OsNRAMP7, TBR225 rice
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