CRISPR/Cas9介导的多基因编辑系统在大豆中的应用

Application of CRISPR/Cas9-mediated multiplex genome editing system in soybean genome editing

自CRISPR/Cas9(clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9)基因组编辑技术发现以来,迅速在作物中得到广泛应用。但是,CRISPR/Cas9多基因编辑系统在大豆中的研究尚待开发。本文利用CRISPR/Cas9介导的多基因编辑系统,分别构建了两个载体,一个载体含6个靶点,编辑7个大豆基因(4个Glycine max ASYMMETRIC LEAVES1(GmAS1)同源基因和3个GmAS2同源基因),另一个载体含8个靶点,编辑11个G.max AGAMOUS家族同源基因(4个GmAG同源基因,2个G.max SEEDSTICK(GmSTK)同源基因和5个G.max SHATTERPROOF1(GmSHP1/2)同源基因)。大豆遗传转化后,经表型鉴定和靶点检测发现,CRISPR/Cas9介导的多基因编辑系统在大豆中成功实现了多基因编辑。当3个GmAS1同源基因和3个GmAS2同源基因同时突变时,导致大豆叶片向远轴面弯曲、皱缩且叶柄变短的表型。当2个GmSHP1同源基因和2个GmSTK同源基因同时突变时,导致豆荚停止发育的不育表型。

To apply CRISPR/Cas9(clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9)based genome editing system on soybean functional genomics and breeding,CRISPR/Cas9-mediated targeted mutagenesis were obtained for more than 5 genes in soybean.One CRISPR/Cas9 plasmid conferred 6 sgRNA expression cassettes targeting 4 Glycine max ASYMMETRIC LEAVES1(GmAS1)genes and 3 GmAS2 genes,and another plasmid conferred 8 sgRNA expression cassettes targeting 6 G.max AGAMOUS1(GmAG)genes and 5 G.max SHATTERPROOF1(GmSHP1/2)genes.From the double mutagenesis of 3 GmAS1 genes and 3 GmAS2 genes,a phenotype of upward curling leaves and shorten leaf petiole length was discovered.From the double mutagenesis of 2 GmSHP1/2 genes and 2 GmSTK genes,sterility phenotype was found.