CRISPR/Cas9 ribonucleoprotein-mediated knockout of Gly m 4-L1 eliminates allergen accumulation in soybean

Gly m 4 is a major allergen in soy milk and cross-reacts with Bet v 1, the primary birch pollen allergen. To reduce Gly m 4 allergenicity, we performed site-directed mutagenesis of the Gly m 4 gene in soybean through a DNA-free in planta bombardment method using ribonucleoproteins (iPB-RNP). Although a mutant line (Gly m 4-2^del ) was generated by targeting the Gly m 4-2 gene, immunoblot analysis revealed that translation products from other homologs still accumulated in the seed tissues. Subsequent gene expression analysis identified Gly m 4-L1, one of eight Gly m 4 homologs in the soybean genome, as the primary target for further mutagenesis. Ribonucleoprotein complexes loaded with either a single guide RNA (gRNA) or two distinct gRNAs targeting Gly m 4-L1 were introduced into the shoot apical meristems. Sequencing analysis identified three mutant lines: an 8-bp deletion (Gly m 4-L1^8-del ), a 128-bp insertion (Gly m 4-L1^128-ins ), and a complete gene deletion (Gly m 4-L1^null ). Immunoblot analysis confirmed the absence of Gly m 4 protein accumulation in the seeds of these mutants. To evaluate immunoglobulin E (IgE) reactivity, protein extracts from Gly m 4-2^del , Gly m 4-L1^null , and wild-type plants were incubated with sera from patients positive for Gly m 4-specific IgE. Protein extracts from the Gly m 4-L1^null line showed markedly reduced IgE binding compared with Gly m 4-2^del and wild-type samples. These findings demonstrate that Gly m 4-L1 is the key gene responsible for Gly m 4 allergen production in soybean seeds.