CRISPR/Cas9 editing of β-Conglycinin subunits reduces IgE binding in soybean [Glycine max (L.) Merr.]

Soybean [Glycine max (L.) Merr.] is a major source of plant-based protein, yet the seed storage protein β-conglycinin (7 S globulin) is a prominent allergen. The αʹ, α, and β subunits contain IgE-binding epitopes, and their high sequence similarity enables simultaneous genome editing. The development of soybean lines with reduced β-conglycinin-specific IgE-binding capacity could enhance food safety for individuals with soy allergies. We employed CRISPR/Cas9 to disrupt the αʹ (Glyma.10G246300) and α (Glyma.20G148300, Glyma.20G148400), subunit genes and to target the β subunit genes (Glyma.20G146200, Glyma.20G148200) of β-conglycinin, generating four edited lines: SP1 (αʹ-null), SP2 (αʹα-null), SP3 (β-null), and SP4, which shows an αʹα-edited genotype and a β subunit-null protein phenotype. SDS-PAGE and DNA sequencing confirmed complete or near-complete loss of the targeted proteins across the T0 to T6 generations, demonstrating stable inheritance of the edited seed protein profiles. IgE immunoblotting and inhibition ELISA using pooled sera from soy-allergic individuals revealed distinct IgE-binding inhibition profiles among the edited lines. At the highest inhibitor concentration, SP4 showed the lowest IgE-binding inhibition (70.0%) compared with the wild type (87.7%), whereas SP1-SP3 exhibited inhibition values similar to or only slightly lower than those of the wild type. CRISPR/Cas9-mediated elimination of β-conglycinin subunits reduces IgE binding to soybean seed proteins and yields lines with stably inherited seed protein phenotypes. These results highlight the potential of targeted genome editing to generate soybean lines with reduced β-conglycinin-specific IgE recognition, supporting the application of precise genome modification in crop improvement for safer soy-based foods.