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 (T₀ plants) were regenerated from two of the transgenic embryo lines. The segregation pattern of the Cas9 gene in the T₁ 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 T₀ plants, we could remove foreign DNA easily by genetic segregation in the T₁ generation. Our results demonstrate that biolistic transformation of soybean embryos is useful for CRISPR/Cas9-mediated site-directed mutagenesis of soybean for human consumption.

成簇规则间隔短回文重复序列 (CRISPR)/CRISPR 相关核酸内切酶 9 (Cas9) 系统正在快速开发用于高等植物的诱变。理想情况下，该系统引入的外源 DNA 在食用作物和蔬菜的育种过程中会被去除。在这里，我们报告了使用基因枪转化和 CRISPR/Cas9 系统高效生成缺乏过敏基因Gly m Bd 30K的无Cas9突变体。根据潮霉素抗性选择了五个转基因胚胎系。切割扩增多态性序列分析仅在所有品系中检测到两种不同的突变。这些结果表明，在将外源基因递送至胚胎细胞后，立即在靶基因中诱导突变。大豆幼苗（T ₀植物）由两个转基因胚系再生。 T ₁代（包括无Cas9植物）中Cas9基因的分离模式表明，两个品系中都整合了单拷贝数的转基因。免疫印迹分析表明，在无Cas9的植物中没有积累Gly m Bd 30K蛋白。基因表达分析表明，成熟突变种子中可能发生了无义 mRNA 衰减。由于T ₀植物中可遗传突变的有效诱导和低整合转基因拷贝数，我们可以通过T ₁代中的遗传分离轻松去除外源DNA。我们的结果表明，大豆胚胎的基因枪转化可用于 CRISPR/Cas9 介导的人类食用大豆定点诱变。