CRISPR-Cas and RNA sequencing reveal nutrient enhancement pathways in quinoa for plant-based athlete recovery diets and future food security.

Quinoa (Chenopodium quinoa Willd.) is a nutrient-dense, climate-resilient pseudocereal with growing relevance to food and nutritional security. However, its potential remains underutilized due to suboptimal levels of key micronutrients. In this study, to apply CRISPR-Cas9 for the simultaneous enhancement of multiple nutrients in quinoa, marking a significant advancement in crop biofortification, we employed multiplexed CRISPR-Cas9 genome editing to target five genes involved in lysine transport, phytic acid biosynthesis, and vitamin C and E biosynthetic pathways. Both knockout and homology-directed knock-in strategies were applied to induce heritable mutations in genes such as CqAAP1, CqIPK1, CqGGP, and CqHPT. Homology-directed knock-in refers to a precise gene-editing method that uses a template to insert specific genetic changes at targeted sites in the genome. Edited lines exhibited significant improvements in seed nutrient concentrations, including lysine (+35 %), zinc (+43 %), vitamin C (+114 %), and vitamin E (+45 %), without yield or growth penalties. Whole-transcriptome profiling via RNA sequencing (RNA-Seq) identified 1284 differentially expressed genes (FDR < 0.05), predominantly associated with amino acid metabolism, redox regulation, and vitamin biosynthesis. Gene Ontology (GO) and KEGG enrichment analyses confirmed the transcriptional activation of nutrient assimilation and antioxidant pathways. Integration of qRT-PCR with RNA-Seq confirmed the reliability of expression data. Structural validations of edits were performed using PCR, gel electrophoresis, and Sanger sequencing. Phenotypic assessments, including seed weight, morphology, and exploratory herbivory assays, confirmed agronomic stability. This study presents a technically validated framework for simultaneous nutritional trait stacking in quinoa using nucleic acid engineering tools. Our findings offer critical insights into the transcriptional plasticity of quinoa and establish a functional genomics platform for developing multi-nutrient biofortified crops to address hidden hunger and advance sustainable food systems.