Precise knock‐in of stress‐responsive <i>cis</i>‐regulatory elements using gene targeting for improving abiotic stress tolerance in plants

Plants are subjected to a multitude of biotic and abiotic stresses, which frequently impede growth and may result in economic losses. One of the primary objectives in plant breeding is the development of stress-tolerant varieties. The modification of protein-coding sequences through gene editing and transgenic methods is a widely utilized approach for enhancing stress tolerance in plants. By contrast, the manipulation of promoter sequences to optimize their expression in a precise temporal and spatial manner has rarely been employed. This study employs the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-Associated Protein 9-mediated gene targeting (GT) method for the precise knock-in (KI) of stress-responsive cis-acting regulatory elements (SRCEs) into the promoter region of candidate genes in Arabidopsis. The SRCE-KI plants demonstrated enhanced tolerance to abiotic stresses without any adverse effects. The SRCE-KI plants exhibited rapid stomatal closure, a reduced rate of leaf water loss, and diminished oxidative damage under stress conditions. These findings suggest that GT is a promising genome manipulation technology and that manipulation of transcriptional regulatory regions may be an effective strategy for enhancing plant traits. The findings of this study provide new insights into the potential of in-locus promoter manipulation of candidate genes as a novel approach in plant research and molecular breeding.