Site-directed mutagenesis by biolistic transformation efficiently generates inheritable mutations in a targeted locus in soybean somatic embryos and transgene-free descendants in the T-1 generation

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system is being rapidly developed for mutagenesis in higher plants. Ideally, foreign DNA introduced by this system is removed in the breeding of edible crops and vegetables. Here, we report an efficient generation of Cas9-free mutants lacking an allergenic gene, Gly m Bd 30K, using biolistic transformation and the CRISPR/Cas9 system. Five transgenic embryo lines were selected on the basis of hygromycin resistance. Cleaved amplified polymorphic sequence analysis detected only two different mutations in e all of the lines. These results indicate that mutations were induced in the target gene immediately after the delivery of the exogenous gene into the embryo cells. Soybean plantlets (T-0 plants) were regenerated from two of the transgenic embryo lines. The segregation pattern of the Cas9 gene in the T-1 generation, which included Cas9-free plants, revealed that a single copy number of transgene was integrated in both lines. Immunoblot analysis demonstrated that no Gly m Bd 30K protein accumulated in the Cas9-free plants. Gene expression analysis indicated that nonsense mRNA decay might have occurred in mature mutant seeds. Due to the efficient induction of inheritable mutations and the low integrated transgene copy number in the T-0 plants, we could remove foreign DNA easily by genetic segregation in the T-1 generation. Our results demonstrate that biolistic transformation of soybean embryos is useful for CRISPR/Cas9-mediated site-directed mutagenesis of soybean for human consumption

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 T-0 Enrei plants and nine T-0 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 T-2 and T-3 generations. Variable mutational spectra were observed in the targeted loci even in T-2 and T-3 progenies of the same T-0 plant. Induction of multiple mutant alleles resulted in six haplotypes in the Cas9-free mutants derived from one T-0 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