利用CRISPR/Cas9技术靶向编辑水稻基因

Application of the technology of CRISPR/Cas9 edit rice gene

以现有水稻CRISPR/Cas9载体系统为起点,优化改造了相应的载体及其构建方法,实现了两步法构建基因敲除载体。利用新的载体和方法,针对水稻日本晴(Oryza sativa L.spp.Japonica cv.Nipponbare)D3基因的3个靶向位点分别构建了相应的CRISPR/Cas9载体,并通过农杆菌介导的遗传转化成功获得一系列转基因植株。T_0代转基因植株靶位点测序结果显示,靶位点DDRC1包括5种突变类型,植株的突变率为35.48%;靶位点DDRC2包括5种突变类型,植株的突变率为25.00%;靶位点DDRC3包括1种突变类型,植株的突变率为20.00%。利用T_1代纯合突变植株,进一步研究了所得纯合突变体的株高、分蘖数表型与CRISPR/Cas9编辑后基因型之间的关系,探讨了CRISPR/Cas9系统的基因编辑效率、位置效应和突变类型等特点,为进一步利用CRISPR/Cas9系统发展具有不同农艺性状的水稻种质材料提供了参考。

In this study,the CRISPR/Cas9vector system was optimized in order to construct gene editing constructors in two steps.With optimized CRISPR/Cas9system,three genome editing constructors targeting different sites of D3gene in rice(Oryza sativa L.spp.Japonica cv.Nipponbare)were generated,and the transgenic plants were successfully produced by Agrobacteriummediated transformation.The sequencing results of T0transgenic plants showed that the mutation rate of target site DDRC1was35.48%,and five mutation types were detected;the mutation rate of target site DDRC2was25.00%,and also with five mutation types;the mutation rate of target site DDRC3was20.00%,and only one mutation type was detected.The phenotype,height and tiller number,together with the genotypes of homozygous mutants were investigated,and the gene editing efficiency of the optimized CRISPR/Cas9system along with the position effect and mutation patterns were also analyzed and discussed.In summary,the findings in this research provided important information for further developing and utilizing of rice germplasm on variety agronomic trait with the optimized CRISPR/Cas9system.