Wang Longxiang, Wang Longlong, Tan Qian, Fan Qiuling, Zhu Hui, Hong Zonglie, Zhang Zhongming, Duanmu Deqiang
State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University Wuhan, China.
College of Life Science and Technology, Huazhong Agricultural University Wuhan, China.
Front Plant Sci. 2016 Aug 31;7:1333. doi: 10.3389/fpls.2016.01333. eCollection 2016.
The targeted genome editing technique, CRISPR/Cas9 system, has been widely used to modify genes of interest in a predictable and precise manner. In this study, we describe the CRISPR/Cas9-mediated efficient editing of representative SNF (symbiotic nitrogen fixation) related genes in the model legume Lotus japonicus via Agrobacterium-mediated stable or hairy root transformation. We first predicted nine endogenous U6 genes in Lotus and then demonstrated the efficacy of the LjU6-1 gene promoter in driving expression of single guide RNAs (sgRNAs) by using a split yellow fluorescence protein (YFP) reporter system to restore the fluorescence in Arabidopsis protoplasts. Next, we chose a customized sgRNA targeting SYMRK (symbiosis receptor-like kinase) loci and achieved ~35% mutagenic efficiency in 20 T0 transgenic plants, two of them containing biallelic homozygous mutations with a 2-bp deletion near the PAM region. We further designed two sgRNAs targeting three homologous leghemoglobin loci (LjLb1, LjLb2, LjLb3) for testing the possibility of generating multi-gene knockouts. 20 out of 70 hairy root transgenic plants exhibited white nodules, with at least two LjLbs disrupted in each plant. Compared with the constitutively active CaMV 35S promoter, the nodule-specific LjLb2 promoter was also effective in gene editing in nodules by hairy root transformation. Triple mutant knockout of LjLbs was also obtained by stable transformation using two sgRNAs. Collectively, these studies demonstrate that the CRISPR/Cas9 system should greatly facilitate functional analyses of SNF related genes in Lotus japonicus.
靶向基因组编辑技术CRISPR/Cas9系统已被广泛用于以可预测且精确的方式修饰感兴趣的基因。在本研究中，我们描述了通过农杆菌介导的稳定或毛状根转化，利用CRISPR/Cas9系统对模式豆科植物百脉根中具有代表性的共生固氮（SNF）相关基因进行高效编辑。我们首先预测了百脉根中的9个内源性U6基因，然后通过使用分裂型黄色荧光蛋白（YFP）报告系统在拟南芥原生质体中恢复荧光，证明了LjU6-1基因启动子驱动单向导RNA（sgRNA）表达的效力。接下来，我们选择了一个靶向SYMRK（共生受体样激酶）位点的定制sgRNA，并在20株T0转基因植物中实现了约35%的诱变效率，其中两株在PAM区域附近有一个2 bp缺失的双等位基因纯合突变。我们进一步设计了两个靶向三个同源豆血红蛋白基因座（LjLb1、LjLb2、LjLb3）的sgRNA，以测试产生多基因敲除的可能性。70株毛状根转基因植物中有20株表现出白色根瘤，每株植物中至少有两个LjLb基因被破坏。与组成型活性CaMV 35S启动子相比，根瘤特异性LjLb2启动子通过毛状根转化在根瘤基因编辑中也有效。通过使用两个sgRNA进行稳定转化，也获得了LjLb基因的三突变敲除植株。总的来说，这些研究表明CRISPR/Cas9系统将极大地促进百脉根中SNF相关基因的功能分析。
