CRISPR-Cas9-driven improvement of foxtail millet (Setaria italica) for enhanced nutraceutical potential: Metabolomic profiling, efficacy, and safety evaluation

Millets are significant components of the Poaceae (Gramineae) family and are thus valued for their adaptability and also endurance, particularly in the dry portions of Asia and Africa Scientific research on plants and agriculture has undergone radical transformations thanks to the CRISPR/Cas9-based genome editing toolset. A very effective genome editing system for foxtail millet (Setaria italica) is needed in order to build durable genetic transformation protocols. Foxtail millet is usually valued for its content of lysine, which is an indispensable amino acid that is often absent in other cereals. This makes it a crucial source of supplementary protein, mostly in the regions where protein deficiencies are predominant. Its addition in the diet not only upsurges the intake of protein but also augments the overall the nutritious value of other grains. Phytic acid (PA), also identified as Myo-inositol-1, 2, 3, 4, 5, 6 hexakisphosphate (IP₆), is thought to be the main storage form of nutrient phosphorous (P) Due to the lack of phytase, monogastric animals cannot utilize PA and the micronutrients in phytates as a nutrient source . This study combines in-silico analysis, CRISPR-Cas9 based genome editing, metabolomics, antioxidant bioactivity and molecular docking to study the possibility of developing nutritionally enriched and functionally improved Indian foxtail millet (Setaria italica) with reduced phytic acid content. The key gene of phytic acid biosynthetic pathway, inositol polyphosphate kinase (IPK) gene was studied to understand the regulatory and functional characteristics. Promoter analysis was carried out to identify potential cis-regulatory elements and transcription factor binding sites that might affect the expression of IPK genes. This study also involves the metabolomics analysis by GC/MS technique which offer opportunities for genome editing to develop low phytic acid trait in Indian Millet. Integration of CRISPR-Cas9 genome editing with metabolomic profiling therefore provides a powerful framework for the development of low-phytic-acid foxtail millet with improved nutritional and functional properties. The produced molecular and metabolomic information may also provide the basis for future quality control and regulatory assessment of genome edited millet based products.