油菜角果的多室特性是培育高产油菜最重要的目标农艺性状之一。目前，关于油菜角果多室性状的自然或人工诱变尚未见报道，这主要是由于油菜的双二倍体特性以及多拷贝的功能冗余基因突变的概率较低。近日，华中农业大学的范楚川团队利用CRISPR/Cas9系统成功敲除油菜CLV3同源基因以及CLV信号通路中相关受体基因CLV1 和CLV2，并实现在三个世代中稳定遗传。在油菜的两个亚基因组中每个BnCLV基因有两个拷贝。只有当每个BnCLV基因的两个拷贝都被敲除时，才能获得角果的多室表型，表明通过CRISPR/Cas9技术同时突变多拷贝的功能基因能够有效突变油菜控制重要农艺性状的基因。不同sgRNAs介导的T0代植株的突变效率为0%-48.65%，表明优选的sgRNA对于高效的油菜基因编辑至关重要。进一步研究发现，与野生型和单突变植株相比，BnCLV3基因的双突变植株表现为叶片增多，每个角果的种子粒数提高、种子重量增加，因而具有很高的增产潜力。此外，该研究还评价了Cas9/gRNA表达盒通过花粉进行水平转移的效率。该研究为基因编辑技术应用于油菜遗传改良提供了范例，相关内容发表在Plant Biotechnol J上。
Plant Biotechnol J, 18 December 2017
Precise editing of CLAVATA genes in Brassica napus L. regulates multilocular silique development
Yang Yang, Kaiyu Zhu, Huailin Li, Shaoqing Han, Qingwei Meng, Shahid Ullah Khan, Chuchuan Fan, Kabin Xie, Yongming Zhou
Multilocular silique is a desirable agricultural trait with great potential for the development of high-yield varieties of Brassica. To date, no spontaneous or induced multilocular mutants have been reported in Brassica napus, which likely reflects its allotetraploid nature and the extremely low probability of the simultaneous random mutagenesis of multiple gene copies with functional redundancy. Here, we present evidence for the efficient knockout of rapeseed homologues ofCLAVATA3 (CLV3) for a secreted peptide and its related receptors CLV1 and CLV2 in the CLV signalling pathway using the CRISPR/Cas9 system and achieved stable transmission of the mutations across three generations. Each BnCLV gene has two copies located in two sub-genomes. The multilocular phenotype can be recovered only in knockout mutations of both copies of each BnCLV gene, illustrating that the simultaneous alteration of multiple gene copies by CRISPR/Cas9 mutagenesis has great potential in generating agronomically important mutations in rapeseed. The mutagenesis efficiency varied widely from 0% to 48.65% in T0 with different single-guide RNAs (sgRNAs), indicating that the appropriate selection of the sgRNA is important for effectively generating indels in rapeseed. The double mutation of BnCLV3 produced more leaves and multilocular siliques with a significantly higher number of seeds per silique and a higher seed weight than the wild-type and single mutant plants, potentially contributing to increased seed production. We also assessed the efficiency of the horizontal transfer of Cas9/gRNA cassettes by pollination. Our findings reveal the potential for plant breeding strategies to improve yield traits in currently cultivated rapeseed varieties.