Study of Qualitative and Quantitative Detection Methods for Exogenous Gene Cas9 in Gene Editing
DING Lin1,*, WANG Hao-Qian2,*, ZHANG Min1, WANG Yu-Ling1, WANG Xiao-Fu1, CHEN Xiao-Yun1, XU Jun-Feng1, ZHANG Xiu-Jie2,**, PENG Cheng1,**
1 Institute of Agro-product Safety and Nutrition, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China; 2 Development Center for Science and Technology, Ministry of Agriculture and Rural Affairs of the People's Republic of China, Beijing 100176,China
Abstract：The prerequisites of bringing gene-edited products to market are effective supervision of them and eliminating public doubts in China. At present, Cas9 endonuclease is the most commonly used method of gene-editing technology, therefore, it is necessary to establish a qualitative and quantitative detection method for Cas9 endonuclease. In this study, the most commonly used Cas9 endonuclease exogenous sequence in gene-editing was used as the target site. The primers and probes of conventional PCR and qPCR were designed, and the most suitable primers and probes were selected through experiments. The system was optimized in the conventional PCR method to determine the optimal primer concentration and annealing temperature for subsequent experiments. By testing specificity and sensitivity, conventional PCR and qPCR detection methods were established for detecting the Cas9 in gene-editing. Meanwhile, the detection limits of qualitative and quantitative detection methods were determined. In the specificity experiment of detection method, the target band was amplified by conventional PCR method in the samples containing Cas9, but the samples without Cas9 endonuclease were negative results. The qPCR method obtained amplification curves in samples containing Cas9, while other samples without Cas9 endonuclease were not amplified; In the method sensitivity test, conventional PCR could detect 0.1% of Cas9 event, and limit of detection (LOD) of qPCR method could reach 16 copies of gene-edited rice genomic DNA; In the method stability experiment, the conventional PCR performed 67 repeated experiments by using the gene-edited samples with a mass fraction of 0.1%, and the experimental results all amplified the target bands. The qPCR method was repeated 60 times at the corresponding minimum detection limit concentration, and the experimental results all obtained the amplification curve. At the same time, this qualitative and quantitative method is applicable to different gene-edited crops, such as rice (Oryza sativa), soybean (Glycine max) and rape (Brassica napus). The qualitative and quantitative detection methods of Cas9 established in this study have good specificity, high sensitivity and strong stability. The establishment of this method will provide a certain technical support for the effective supervision of gene-edited products in the future.
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