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<p>Pre-harvest sprouting (PHS), where seeds germinate on panicles before harvest under humid conditions, is a serious global issue in cereal crop production, including rice. Fine-mapping of the previously reported chromosome 4 locus identified <i>OsERF94</i> as a strong candidate gene for functional validation. In this study, we investigated the role of <i>OsERF94</i> in PHS using CRISPR/Cas9 gene editing. The CRISPR/Cas9-mediated mutagenesis of <i>OsERF94</i> induced frameshift mutations, resulting in a loss-of-function of <i>OsERF94</i> in the 1-I-ET and 2-D-ET lines. The 1-I-ET and 2-D-ET lines exhibited significantly higher germination rates under PHS conditions compared to the wild type, indicating increased susceptibility to PHS. Whole-genome re-sequencing confirmed that few or no mutations could be detected at off-target candidate sites in both edited lines, ensuring the precision of the CRISPR/Cas9 gene editing. A transcriptome analysis revealed altered expression patterns of several GA-related genes, including <i>OsLOL1</i>, <i>OsKO3</i>, <i>OsGA3ox2</i>, and <i>OsGA2ox5</i> in the <i>OsERF94</i> mutant lines. The up-regulation of GA biosynthetic genes and the down-regulation of GA deactivation genes observed in both the <i>OsERF94</i> mutant lines suggest possible alterations in GA metabolism during the early stages of PHS. Transient luciferase reporter assays using a single-luciferase system suggested that <i>OsERF94</i> may be associated with changes in the promoter activities of several GA- and ethylene-related genes. These findings suggest that <i>OsERF94</i> may contribute to the regulation of PHS, potentially through moderation of GA- and ethylene-related pathways. Overall, this study improves our understanding of the molecular role of <i>OsERF94</i> in PHS and highlights its potential as a target for the genetic improvement of PHS resistance in rice-breeding programs.</p>

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CRISPR/Cas9-Mediated Mutagenesis of OsERF94 Enhances Pre-Harvest Sprouting in Rice

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年6月27日

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  • Man Bo Lee,
  • Ha Neul Lee,
  • Sang-Ho Chu,
  • Yong-Jin Park,
  • Chang-Wook Park,
  • In-Jung Lee,
  • Lae-Hyeon Cho,
  • Jae Yoon Kim

摘要

Pre-harvest sprouting (PHS), where seeds germinate on panicles before harvest under humid conditions, is a serious global issue in cereal crop production, including rice. Fine-mapping of the previously reported chromosome 4 locus identified OsERF94 as a strong candidate gene for functional validation. In this study, we investigated the role of OsERF94 in PHS using CRISPR/Cas9 gene editing. The CRISPR/Cas9-mediated mutagenesis of OsERF94 induced frameshift mutations, resulting in a loss-of-function of OsERF94 in the 1-I-ET and 2-D-ET lines. The 1-I-ET and 2-D-ET lines exhibited significantly higher germination rates under PHS conditions compared to the wild type, indicating increased susceptibility to PHS. Whole-genome re-sequencing confirmed that few or no mutations could be detected at off-target candidate sites in both edited lines, ensuring the precision of the CRISPR/Cas9 gene editing. A transcriptome analysis revealed altered expression patterns of several GA-related genes, including OsLOL1, OsKO3, OsGA3ox2, and OsGA2ox5 in the OsERF94 mutant lines. The up-regulation of GA biosynthetic genes and the down-regulation of GA deactivation genes observed in both the OsERF94 mutant lines suggest possible alterations in GA metabolism during the early stages of PHS. Transient luciferase reporter assays using a single-luciferase system suggested that OsERF94 may be associated with changes in the promoter activities of several GA- and ethylene-related genes. These findings suggest that OsERF94 may contribute to the regulation of PHS, potentially through moderation of GA- and ethylene-related pathways. Overall, this study improves our understanding of the molecular role of OsERF94 in PHS and highlights its potential as a target for the genetic improvement of PHS resistance in rice-breeding programs.