<title>Abstract</title>
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<title>Background</title>
Soybean (<italic>Glycine max</italic>) is a major protein crop, because soybean protein has an amino acid score comparable to that of beef and egg white. However, many allergens have been identified among soybean proteins. A decrease in allergenic protein levels would be useful for expanding the market for soybean proteins and processed foods. Recently, the CRISPR/Cas9 system has been adopted as a powerful tool for the site-directed mutagenesis in higher plants. This system is expected to generate hypoallergenic soybean varieties.


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<title>Results</title>
We used two guide RNAs (gRNAs) and <italic>Agrobacterium</italic>-mediated transformation for simultaneous site-directed mutagenesis of two genes encoding the major allergens Gly m Bd 28 K and Gly m Bd 30 K in two Japanese soybean varieties, Enrei and Kariyutaka. We obtained two independent T₀ Enrei plants and nine T₀ Kariyutaka plants. Cleaved amplified polymorphic sequence (CAPS) analysis revealed that mutations were induced in both targeted loci of both soybean varieties. Sequencing analysis showed that deletions were the predominant mutation type in the targeted loci. The <italic>Cas9</italic>-free plants carrying the mutant alleles of the targeted loci with the transgenes excluded by genetic segregation were obtained in the T₂ and T₃ generations. Variable mutational spectra were observed in the targeted loci even in T₂ and T₃ progenies of the same T₀ plant. Induction of multiple mutant alleles resulted in six haplotypes in the <italic>Cas9</italic>-free mutants derived from one T₀ plant. Immunoblot analysis revealed that no Gly m Bd 28 K or Gly m Bd 30 K protein accumulated in the seeds of the <italic>Cas9</italic>-free plants. Whole-genome sequencing confirmed that a <italic>Cas9</italic>-free mutant had also no the other foreign DNA from the binary vector. Our results demonstrate the applicability of the CRISPR/Cas9 system for the production of hypoallergenic soybean plants.


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<title>Conclusions</title>
Simultaneous site-directed mutagenesis by the CRISPR/Cas9 system removed two major allergenic proteins from mature soybean seeds. This system enables rapid and efficient modification of seed components in soybean varieties.


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