CRISPR/Cas9 targeted mutagenesis of OsTRE1 rice gene generates lines with improved physiological responses to salinity under in vitro conditions

Abiotic stresses such as osmotic stress and salinity severely limit rice productivity, while developing stress-tolerant varieties remains a significant challenge. Although trehalose metabolism has been linked to stress adaptation, targeted genome editing of the trehalase gene in rice has not been thoroughly explored. This study uses the CRISPR/Cas9 system to edit the trehalase gene (OsTRE1) in Oryza sativa L. subsp. japonica cv. Nipponbare to evaluate its potential to enhance abiotic stress tolerance. Two sgRNAs targeting different sites within exon 1 were designed, and the CRISPR/Cas9 construct was introduced into embryogenic calli through Agrobacterium tumefaciens-mediated transformation. A total of 15 T0 transgenic lines were obtained, of which six (40%) carried mutations in exon 1. Among these mutants, five (83.3%) were biallelic, displaying insertions, deletions, and substitutions at the target sites, while one mutant (16.7%) was homozygous with two deletions in exon 1. In silico analysis of the predicted amino acid sequences revealed two cases in which editing resulted in the substitution or loss of one or two amino acids. In contrast, most mutations caused large deletions of 98–122 amino acids at the protein N-terminus. T1 targeted-mutagenized lines were evaluated in vitro for tolerance to salinity (200 mM NaCl) and osmotic stress (15% w/v sorbitol). No line showed tolerance to sorbitol; however, several targeted-mutagenized lines exhibited improved salinity tolerance compared with the wild type (WT). Overall, this study provides the first evidence of targeted OsTRE1 mutagenesis in japonica rice and demonstrates that disrupting trehalase function can enhance salinity tolerance. The results obtained in this research support trehalase-focused genome editing as a viable approach for developing salinity-resilient rice varieties. Such varieties may better withstand salinity stress driven by climate change, including seawater intrusion due to sea-level rise or soil salinization caused by reduced precipitation.