Multiplex CRISPR/Cas9 Editing of Rice Prolamin and GluA Glutelin Genes Reveals Subfamily-Specific Effects on Seed Protein Composition

Rice seed storage proteins (SSPs) play a critical role in determining the nutritional quality, cooking properties, and digestibility of rice. To enhance seed quality, CRISPR/Cas9 genome editing was applied to modify SSP composition by targeting genes encoding 13 kDa prolamins and type A glutelins. Three CRISPR/Cas9 constructs were designed: one specific to the 13 kDa prolamin subfamily and two targeting conserved GluA glutelin regions. Edited T₀ and T₁ lines were generated and analyzed using InDel analysis, SDS-PAGE, Bradford assay, and RP-HPLC. Insertions were more frequent than deletions, accounting for 56% and 74% of mutations in prolamin and glutelin genes, respectively. Editing efficiency varied between sgRNAs. All lines with altered protein profiles contained InDels in target genes. SDS-PAGE confirmed the absence or reduction in bands corresponding to 13 kDa prolamins or GluA subunits, showing consistent profiles among lines carrying the same construct. Quantification revealed significant shifts in SSP composition, including increased albumin and globulin content. Prolamin-deficient lines showed reduced prolamins, while GluA-deficient lines exhibited increased prolamins. Total protein content was significantly elevated in all edited lines, suggesting enrichment in lysine-rich fractions. These findings demonstrate that CRISPR/Cas9-mediated editing of SSP genes can effectively reconfigure the rice protein profile and enhance its nutritional value.