It is suggested that OsERF94 may contribute to the regulation of PHS, potentially through moderation of GA- and ethylene-related pathways.
Abstract
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.
Rice (Oryza sativa L.) is a staple food crop worldwide, and improving disease resistance is a core target in rice breeding. In this study, we employed CRISPR/Cas9 genome editing to modify the coding sequence (CDS) of two susceptibility genes, Bsr-d1 and Pi21, in the elite maintainer line Gengxiang B to enhance its blast resistance. We generated Bsr-d1/Pi21 double homozygous mutants via Agrobacterium-mediated genetic transformation. Quantitative RT-PCR revealed significantly suppressed transcript accumulation of both target genes in the edited lines compared with the wild type Gengxiang B. Upon inoculation with Magnaporthe oryzae, multiple defense-related marker genes were markedly upregulated in the double mutants. Phenotypic assays demonstrated significantly reduced disease severity for both leaf and panicle blast in the edited lines compared with the wild type. Importantly, no statistically detectable differences were found between the double mutants and wild-type plants for key agronomic or grain quality traits. Collectively, these results demonstrate that CRISPR/Cas9-mediated editing of susceptibility loci generates genetically stable blast-resistant rice germplasm without compromising agronomic traits or grain quality, providing valuable genetic resources for future rice varietal improvement.
Ke Lan, Lin Yuan, Da-Cheng Zhao et al.· Plants· 0 citations
A complete CRISPR-Cas9-mediated knockout of the BEL5 gene, encoding a transcription factor, is reported, known as one of the key regulators driving tuber formation, and a regulatory role of BEL5 in the timing of tuber onset but, unexpectedly, its dispensability for tuber development in modern cultivated potato is proposed.
Andrea Zounková, Daniele Chirivì, A. Přibylová et al.· bioRxiv· 0 citations
Rice is a major cereal crop for global food and nutritional security and a key target for genetic improvement. CRISPR/Cas9 enables precise genetic modification in crops, but mutation screening remains a technical and economic barrier to broader genome-editing applications. Although several detection methods are available, some require labor-intensive procedures, specialized equipment, high costs, or limited sensitivity to specific mutation types. High-resolution melting (HRM) analysis is an established approach for screening CRISPR/Cas-induced mutations in plants, including rice. Here, we evaluated an adapted HRM workflow combining conventional SYBR Green-based qPCR chemistry with downstream computational analysis to detect CRISPR/Cas9-induced mutations at three rice loci: OsMADS26, OsRAC1, and OsNRT1.1b. The workflow detected insertions, deletions, and base substitutions. Across the three loci, the 1% edited-DNA mixtures showed a slight observable deviation from the wild-type melting profile under the conditions evaluated, although this should not be interpreted as a validated detection threshold. Although the assessment of heterozygous samples was limited by their availability, the results support the potential applicability of the approach for individual sample analysis and expanded sample screening. A customizable R script complemented visual analysis by evaluating melting temperature (Tm) and GCP-derived dissimilarity, supporting sample classification. By combining standard SYBR Green chemistry with an adaptable analysis workflow, the method offers an alternative to dedicated HRM reagents and proprietary platforms. This approach provides a practical and potentially lower-cost option for mutation screening at the evaluated rice loci and may be adapted and validated for other targets and plant species.
Bianca Cristina Carvalho Reis, Céline Georget, Anne-Cécile Meunier et al.· New Biotechnology· 0 citations
A multiplex CRISPR/Cas12a system is established in eggplant accession MEL3, representing, to the authors' knowledge, the first application of this nuclease for genome editing in eggplant and demonstrating the potential of Cas12a for functional genomics, allele engineering, and precision breeding in eggplant.
Marina Martínez-López, Andrea Solana, Andrea Arrones et al.· bioRxiv· 0 citations
CRISPR/Cas9 is currently the most powerful genome editing tool for crop improvement and gene function exploration. However, the mutation efficiency of CRISPR/Cas9 in Brassica napus, a globally important allotetraploid oil crop, so far achieved remains significant potential for further improvement compared to that observed in model species. In this study, we systematically evaluated three key factors influencing editing efficiency: sgRNA promoters, sgRNA structural modifications, and Cas9-driven promoters. First, we compared all sgRNA promoters currently used in B. napus side by side. The average editing efficiency of AtU6–26 was the highest (44.74%), followed by AtU3b (36.18%), AtU6–29 (28.49%), AtU3d (17.73%) and AtU6–1 (0.53%). Second, we demonstrated that enhanced sgRNA (esgRNA) enhanced CRISPR/Cas9 editing efficiency by 52.1% compared to native sgRNA. The combination of AtU6–26 with esgRNA achieved the highest editing efficiency (average 61.4%). Third, we identified three endogenous promoters (pCAB1, pLTP2, and pTCTP) with high expression levels in the hypocotyls and callus, to replace the 35S promoter of Cas9. The CAB1 promoter was the most efficient and significantly enhanced the mutation efficiency by 31.46% over the commonly used 35S promoter. Thus, these optimizations provide valuable strategies for improving CRISPR/Cas9 efficiency in Brassica crops.