CRISPR/Cas9-mediated editing of uORFs in the BnVTC2 facilitates abiotic stress resilience without yield penalty

Upstream open reading frame (uORF)-based genetic engineering has emerged as an excellent strategy for improving agronomic traits of crops. Despite its significant potential, the exploration of CRISPR/Cas-based uORF engineering in many crop species remains unexplored, thereby limiting the application of this approach in genetic innovation of important crops. In this study, we focused on uORF-based genome editing to improve abiotic stresses in Brassica napus. The putative uORFs of BnVTC genes which involved in ascorbic acid (AsA) biosynthesis were selected as potential targets. The AsA contents in leaves, buds, and stems were significantly increased in BnVTC2-uORF-edited mutants. The BnVTC2-uORF-edited mutants exhibited tolerance to environmental stresses, such as low temperature, salinity, and drought. No obvious penalty on yield traits were observed between the BnVTC2-uORF-edited lines and WT. VTC2-uORF sequence was highly conserved across the genus Brassica, coupled with the absence of frameshift mutations in the natural germplasm, which suggested that uORF-targeted gene editing alone could be an effective approach for improving abiotic stresses. This research paves the way for the strategic deployment of CRISPR-based uORF engineering to improve the nutritional profile and abiotic stress resistance of oilseed rape.