Graduate School of Agriculture, Hokkaido University, Kita 9, Nishi 9, Kita-ku, Sapporo, Hokkaido, 060-8589, Japan.
Plant Genome Engineering Research Unit, Institute of Agrobiological Sciences, National Agricultural and Food Research Organization, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602, Japan.
Plant Cell Rep. 2018 Mar;37(3):553-563. doi: 10.1007/s00299-018-2251-3. Epub 2018 Jan 15.
Using a gRNA and Agrobacterium-mediated transformation, we performed simultaneous site-directed mutagenesis of two GmPPD loci in soybean. Mutations in GmPPD loci were confirmed in at least 33% of T seeds. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system is a powerful tool for site-directed mutagenesis in crops. Using a single guide RNA (gRNA) and Agrobacterium-mediated transformation, we performed simultaneous site-directed mutagenesis of two homoeologous loci in soybean (Glycine max), GmPPD1 and GmPPD2, which encode the orthologs of Arabidopsis thaliana PEAPOD (PPD). Most of the T plants had heterozygous and/or chimeric mutations for the targeted loci. The sequencing analysis of T and T generations indicates that putative mutation induced in the T plant is transmitted to the T generation. The inheritable mutation induced in the T plant was also detected. This result indicates that continuous induction of mutations during T plant development increases the occurrence of mutations in germ cells, which ensures the transmission of mutations to the next generation. Simultaneous site-directed mutagenesis in both GmPPD loci was confirmed in at least 33% of T seeds examined. Approximately 19% of double mutants did not contain the Cas9/gRNA expression construct. Double mutants with frameshift mutations in both GmPPD1 and GmPPD2 had dome-shaped trifoliate leaves, extremely twisted pods, and produced few seeds. Taken together, our data indicate that continuous induction of mutations in the whole plant and advancing generations of transgenic plants enable efficient simultaneous site-directed mutagenesis in duplicated loci in soybean.
利用 gRNA 和农杆菌介导的转化，我们对大豆中的两个 GmPPD 基因座进行了同时的定点突变。至少 33%的 T 代种子中确认了 GmPPD 基因座的突变。成簇的规律间隔短回文重复序列（CRISPR）/CRISPR 相关内切酶 9（Cas9）系统是作物定点突变的强大工具。利用单个向导 RNA（gRNA）和农杆菌介导的转化，我们对大豆（Glycine max）中的两个同源基因座 GmPPD1 和 GmPPD2 进行了同时的定点突变，这两个基因座编码拟南芥 PEAPOD（PPD）的同源物。大多数 T 代植物在靶基因座上具有杂合和/或嵌合突变。T 和 T 代的测序分析表明，T 代植物中诱导的潜在突变可传递到 T 代。还检测到 T 代植物中诱导的可遗传突变。这一结果表明，在 T 代植物发育过程中连续诱导突变会增加生殖细胞中突变的发生，从而确保突变传递到下一代。在至少 33%的受检 T 代种子中证实了两个 GmPPD 基因座的同时定点突变。大约 19%的双突变体不含 Cas9/gRNA 表达载体。在 GmPPD1 和 GmPPD2 中都发生移码突变的双突变体具有球形三出复叶、极度扭曲的豆荚和很少的种子。总之，我们的数据表明，在整个植物和转基因植物的后代中连续诱导突变，可实现大豆重复基因座的高效同时定点突变。
