The CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR—associated proteins 9) is simple and highly efficient technology applied to functional studies of genes and genetic crop improvement. In this study, we have demonstrated the utility of green fluorescent protein (GFP) marker to detect the targeting efficiency of gRNAs. As a proof of concept, Glycine max De-Etiolated 1 (GmDET1) gene was chosen and tagged with GFP to rapidly analyze genome editing efficiency of gRNAs. Results showed weaker GFP fluorescence signal in the N. benthamiana leaves co-infiltrated with GmDET1-GFP overexpression (OE) + DET1 gRNA1 constructs as compared to the stronger GFP florescence signal in the leaves co-infiltrated with DET1 gRNA2 and gRNA3 constructs, thus indicating the highest of DET1 gRNA1. These results were further confirmed by the detection of the mutation frequencies through T7 endonuclease (T7E1) assay and sequencing; the highest mutation rate of 38.46% in GmDET1 targeted by DET1 gRNA1 to that of DET1 gRNA2 (7.69%) and gRNA3 (15.38%) was observed. Thus our studies showed “GFP tagging” as the most reliable and rapid method-one can apply to minimize the generation of non-edited transgenic plants resulting from inefficient gRNAs.

中文翻译：

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基于GFP标记的方法通过在本氏烟草中的瞬时表达来分析CRISPR / Cas9-gRNA的基因组编辑效率

CRISPR / Cas9（聚簇的规则间隔的短回文重复序列/ CRISPR相关蛋白9）是一种简单高效的技术，用于基因功能研究和遗传农作物改良。在这项研究中，我们已经证明了绿色荧光蛋白（GFP）标记物可用于检测gRNA的靶向效率。作为概念的证明，选择了大豆最大脱甘氨酸1（GmDET1）基因并用GFP标记，以快速分析gRNA的基因组编辑效率。结果显示，与GmDET1 - GFP过表达（OE）+  DET1共同浸润的本氏烟草叶中的GFP荧光信号较弱。与在与DET1 gRNA2和gRNA3构建体共同浸润的叶子中较强的GFP荧光信号相比，gRNA1构建体更强，因此表明DET1 gRNA1最高。通过T7核酸内切酶（T7E1）分析和测序检测突变频率，进一步证实了这些结果。观察到DET1 gRNA1靶向的GmDET1的最高突变率为38.46％，而DET1 gRNA2（7.69％）和gRNA3（15.38％）的突变率最高。因此，我们的研究表明，“ GFP标记”是最可靠，最快速的方法，一种方法可用于最大程度地减少由低效gRNA引起的未经编辑的转基因植物的产生。