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首页> 外文期刊>Frontiers in Plant Science >Efficient Inactivation of Symbiotic Nitrogen Fixation Related Genes in Lotus japonicus Using CRISPR-Cas9
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Efficient Inactivation of Symbiotic Nitrogen Fixation Related Genes in Lotus japonicus Using CRISPR-Cas9

机译：使用CRISPR-Cas9有效灭活中共生固氮相关基因

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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 .
机译：靶向基因组编辑技术CRISPR / Cas9系统已被广泛用于以可预测的精确方式修饰目标基因。在这项研究中，我们描述了通过农杆菌介导的稳定或毛状根转化，在模型豆科植物日本j中由CRISPR / Cas9介导的代表性SNF（共生氮固定）相关基因的有效编辑。我们首先预测了Lotus中的9个内源性U6基因，然后通过使用分裂的黄色荧光蛋白（YFP）报告系统恢复拟南芥原生质体中的荧光，证明了LjU6-1基因启动子在驱动单个向导RNA（sgRNA）表达中的功效。 。接下来，我们选择了针对SYMRK（共生受体样激酶）基因座的定制sgRNA，并在20种T0转基因植物中实现了约35％的诱变效率，其中两株包含双等位基因纯合突变，并在PAM区附近缺失了2个碱基。我们进一步设计了两个针对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。在70根有毛根转基因植物中，有20株显示出白色结节，每株植物中至少有2个LjLb被破坏。与具有组成型活性的CaMV 35S启动子相比，结节特异性LjLb2启动子在通过根毛转化的结节基因编辑中也很有效。 LjLbs的三突变体敲除也通过使用两个sgRNA的稳定转化而获得。总体而言，这些研究表明，CRISPR / Cas9系统应极大地促进日本莲中SNF相关基因的功能分析。

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