Soybean has a palaeopolyploid genome with nearly 75% of the genes present in multiple copies. Although the CRISPR/Cas9 system has been employed in soybean to generate site-directed mutagenesis, a systematical assessment of mutation efficiency of the CRISPR/Cas9 system for the multiple-copy genes is still urgently needed. Here, we successfully optimize one sgRNA CRISPR/Cas9 system in soybean by testing the efficiency, pattern, specificity of the mutations at multiple loci of GmFAD2 and GmALS. The results showed that simultaneous site-directed mutagenesis of two homoeologous loci by one sgRNA, the mutation frequency in the T0 generation were 64.71% for GmPDS, 60.0% for GmFAD2 and 42.86% for GmALS, respectively. The chimeric and heterozygous mutations were dominant types. Moreover, association of phenotypes with mutation pattern at target loci of GmPDS11 and GmPDS18 could help us further demonstrate that the CRISPR/Cas9 system can efficiently generate target specific mutations at multiple loci using one sgRNA in soybean, albeit with a relatively low transformation efficiency.

大豆具有古多倍体基因组，其中近 75% 的基因以多拷贝形式存在。尽管 CRISPR/Cas9 系统已被用于大豆进行定点诱变，但仍迫切需要对 CRISPR/Cas9 系统对多拷贝基因的突变效率进行系统评估。在这里，我们通过测试GmFAD2和GmALS多个位点突变的效率、模式和特异性，成功优化了大豆中的一个 sgRNA CRISPR/Cas9 系统。结果显示有两个同源基因座是同步定点突变由一个因组，在T0代的突变频率分别为64.71％GmPDS，为60.0％GmFAD2和42.86％GmALS， 分别。嵌合和杂合突变是显性类型。此外，表型与GmPDS11和GmPDS18靶位点突变模式的关联可以帮助我们进一步证明 CRISPR/Cas9 系统可以使用大豆中的一个 sgRNA 在多个位点有效地产生目标特异性突变，尽管转化效率相对较低。