Background Soybean (Glycine max) 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. Results We used two guide RNAs (gRNAs) and Agrobacterium-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 Cas9-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 Cas9-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 Cas9-free plants. Whole-genome sequencing confirmed that a Cas9-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. Conclusions 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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SUMMARYIt is well known that surface-relief dielectric gratings with rectangular profile can be treated by uniform approximation of the equivalent permittivity when the periodicity is very small compared with the wavelength. In optics, this phenomenon is the equivalent anisotropic effects or the form birefringence. When the periodicity is very small, the equivalent anisotropic effects will be shown in indexmodulated gratings. In this paper, the uniform approximation is described for the electromagnetic scattering problem of index-modulated gratings. The scattering properties of dielectric slabs are calculated by transmission-line theory and the equivalent permittivity obtained from our proposed formulation of the uniform approximation. Scattering by index-modulated gratings is analyzed rigorously by matrix eigenvalue calculations using the Fourier expansion method and spatial harmonics expansions. When the periodicity is very small, the results are in good agreement. By investigating the difference between the equivalent permittivity and the numerical values corresponding to the permittivity of the index-modulated gratings, the conditions of applicability of the uniform approximation are shown. The equivalent anisotropic effects of various profiles are compared.