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 <i>Gly m 4</i> gene in soybean through a DNA-free <i>in planta</i> bombardment method using ribonucleoproteins (iPB-RNP). Although a mutant line (<i>Gly m 4-2^del</i> ) was generated by targeting the <i>Gly m 4-2</i> gene, immunoblot analysis revealed that translation products from other homologs still accumulated in the seed tissues. Subsequent gene expression analysis identified <i>Gly m 4-L1</i>, one of eight <i>Gly m 4</i> 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 <i>Gly m 4-L1</i> were introduced into the shoot apical meristems. Sequencing analysis identified three mutant lines: an 8-bp deletion (<i>Gly m 4-L1^8-del</i> ), a 128-bp insertion (<i>Gly m 4-L1^128-ins</i> ), and a complete gene deletion (<i>Gly m 4-L1^null</i> ). 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 <i>Gly m 4-2^del</i> , <i>Gly m 4-L1^null</i> , and wild-type plants were incubated with sera from patients positive for Gly m 4-specific IgE. Protein extracts from the <i>Gly m 4-L1^null</i> line showed markedly reduced IgE binding compared with <i>Gly m 4-2^del</i> and wild-type samples. These findings demonstrate that <i>Gly m 4-L1</i> is the key gene responsible for Gly m 4 allergen production in soybean seeds.