CRISPR/CAS-BASED FUNCTIONAL VALIDATION OF KEY SYMBIOTIC GENES CONTROLLING NITROGEN FIXATION IN SOYBEAN

Abstract

Soybean (Glycine max [L.] Merr.), a cornerstone crop for global protein and oil supply, relies on symbiotic nitrogen fixation (SNF) with rhizobia to meet up to 80% of its nitrogen needs, offering a sustainable alternative to synthetic fertilizers amid environmental concerns. However, SNF efficiency is constrained by genetic factors, prompting the need for precise functional validation of key symbiotic genes. This study synthesizes the application of CRISPR/Cas9 technology for targeted editing and validation of genes such as GmNFR1, GmNFR5, GmNIN, and GmRPG, which regulate Nod factor perception, nodule organogenesis, and infection thread formation. CRISPR-mediated knockouts and base editing have confirmed their roles in enhancing nodule number, nitrogenase activity, and fixation rates (up to 30–50% improvements in edited lines), while overcoming host specificity barriers through symbiosis island modifications. Challenges include off-target effects and regulatory hurdles, addressed via prime editing and multiplex systems. Field trials demonstrate yield gains (10–20%) under low-N conditions, underscoring CRISPR's potential for breeding resilient, high-fixing varieties to reduce fertilizer dependency, mitigate emissions, and bolster food security in nitrogen-limited agroecosystems.