Gene editing-mediated enhancement of carotenoid compounds accumulation in common wheat grains

Common wheat ( Triticum aestivum L.) is a staple food crop for humans, yet it primarily accumulates the non-provitamin A carotenoid lutein and exhibits limited natural variation in provitamin A β-carotene among its various accessions. This characteristic necessitates the development of alternative strategies for provitamin A biofortification. To address this challenge, we targeted key control points in the carotenoid biosynthetic pathway using the CRISPR-Cas9 system in a wheat cultivar Fielder. Specifically, we knocked out the gene encoding lycopene ε-cyclase (LCYE), an enzyme that acts as a gatekeeper opposing the production of β-branch carotenoids. This genetic modification resulted in a significant increase in β-carotene levels in the endosperms at 30 DPA of triple homozygous transgene-free mutant lines revealed by biochemical profiling, an approximate 34.5% enhancement for β-carotene, 125.4% for zeaxanthin, 73.8% for violaxanthin, and 186.5% for antheraxanthin compared to the wild-type control. Despite the drastic reduction in lutein levels, the TaLcye mutations did not significantly impair wheat yield and the mutant lines exhibited elevated levels of amylose and soluble sugar. Additionally, the seed coats and endosperms of the triple homozygous transgene-free mutant lines exhibited an orange-yellow hue. In conclusion, we have successfully developed novel carotenoids biofortified wheat lines through gene-editing approach. Our findings demonstrate the potential of gene editing to significantly enhance the nutritional profile of commercial wheat by increasing carotenoid content, thereby addressing micronutrient deficiencies in modern diets.