Simultaneous site-directed mutagenesis for soybean ß-amyrin synthase genes via DNA-free CRISPR/Cas9 system using a single gRNA.

key messageWe generated soybean mutants related to two ß-amyrin synthase genes using DNA-free site-directed mutagenesis system. Our results suggested that one of the genes is predominant in the soyasaponin biosynthesis. Soyasaponins, which are triterpenoid saponins contained in soybean [Glycine max (L.) Merril], are responsible for the astringent aftertaste of soyfood, and their complete elimination from soybean seeds is a key challenge in the development of cultivars with improved taste. While the loss of function in the ß-amyrin synthase genes (GmBAS1 and GmBAS2) has proven effective in reducing soyasaponin content in soybean seeds, the specific functional roles of these genes remain unclear. In this study, site-directed mutagenesis was performed on two GmBAS loci using a DNA-free clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system. A complex of sgRNA targeting sequences conserved in the two loci and Cas9 protein was introduced into the shoot apical meristems of soybean embryonic axes via bombardment. Cleaved amplified polymorphic sequences (CAPS) analysis conducted 1 month post-bombardment revealed that 138 seedlings out of 1,467 screened exhibited mutations at one or both GmBAS loci. CAPS and sequencing analysis in the subsequent generation identified a total of 16 plants with inheritable mutations ranging from one to ten nucleotides. High-performance liquid chromatography (HPLC) analysis showed that site-directed mutagenesis in the GmBAS1 locus resulted in the absence of soyasaponins in mature seeds, as well as in young roots, stems, and leaves. These findings demonstrate that GmBAS1 is the predominant ß-amyrin synthase gene in soybean plants. In addition, the DNA-free CRISPR/Cas9 system was shown to be highly efficient in inducing simultaneous mutagenesis at two target loci using a single gRNA.