Simultaneous induction of mutant alleles of two allergenic genes in soybean by using site-directed mutagenesis.

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.

Recent advances in the improvement of soybean seed traits by genome editing.

Genetic improvement of soybean seed traits is important for developing new varieties that meet the demand for soybean as a food, forage crop, and industrial products. A large number of soybean genome sequences are currently publicly available. This genome sequence information provides a significant opportunity to design genomic approaches to improve soybean traits. Genome editing represents a major advancement in biotechnology. The production of soybean mutants through genome editing is commonly achieved with either an Agrobacterium-mediated or biolistic transformation platform, which have been optimized for various soybean genotypes. Currently, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system, which represents a major advance in genome editing, is used to improve soybean traits, such as fatty acid composition, protein content and composition, flavor, digestibility, size, and seed-coat color. In this review, we summarize the recent advances in the improvement of soybean seed traits through genome editing. We also discuss the characteristics of genome editing using the CRISPR/Cas9 system with transformation platforms.