烟草香气相关基因CRISPR/Cas9编辑突变体库的构建

为探究CRISPR/Cas9基因编辑技术构建烟草突变体库的可行性,该研究以烤烟品种‘红花大金元''为实验材料筛选了100个可能参与烟草香气代谢的基因,设计相应的100个sgRNA并构建了由100个CRISPR/Cas9编辑载体组成的质粒库,获得转基因材料后分析了载体的共转化率、靶向编辑率和脱靶编辑情况。结果表明:(1)通过农杆菌介导100个sgRNA的共转化后,在172个阳性转化株中检测到了其中的77个sgRNA,共转化率为77%。(2)在77个携带sgRNA的转基因后代中,69个sgRNA对目标基因进行了靶向编辑,编辑率为89.6%。(3)脱靶位点测序检测发现,只有1个sgRNA在非目标靶位点产生了脱靶编辑,表明CRISPR/Cas9基因编辑技术在烟草中的脱靶概率非常低。综上所述,利用CRISPR/Cas9载体库共转化对烟草基因进行高通量靶向编辑以构建突变体库的方法切实可行,并且该方法有共转化率高、编辑率高和脱靶编辑概率低等特点。

Construction of a mutant library associated with aroma genes in tobacco using CRISPR/Cas9

To explore the feasibility of constructing a tobacco mutant library using the CRISPR/Cas9 gene editing technology, we designed sgRNAs of 100 aroma related genes in tobacco, constructed a plasmid library composed of 100 corresponding CRISPR/Cas9 editing vectors, and analyzed the co-transformation rate, on-target editing rate, and off-target editing of transgenic offspring using the tobacco variety ‘Honghua Dajinyuan'' as experimental material in this study. The results were as follows:(1)After co-transformation of 100 sgRNAs mediated by Agrobacterium tumefaciens, 77 of them were detected in 172 positive transformation strains, with a co-transformation rate of 77%.(2)Among 77 transgenic offspring carrying sgRNA, 69 sgRNAs edited the target genes, with an editing rate of 89.6%.(3)Sequencing detection revealed that only one sgRNA produced off-target editing at a non-target site, indicating a very low probability of off-target editing of CRISPR/Cas9 in tobacco. In summary, it is feasible to construct a mutant library by the co-transformation of a CRISPR/Cas9 vector library to edit a large number of candidate target genes in tobacco. This method has the characteristics of high co-transformation rate, high editing rate, and low probability of off-target editing.