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Accurate Detection and Evaluation of the Gene-Editing Frequency in Plants Using Droplet Digital PCR

文献类型： 外文期刊

作者： Peng, Cheng 1 ; Zheng, Ming 2 ; Ding, Lin 1 ; Chen, Xiaoyun 1 ; Wang, Xiaofu 1 ; Feng, Xuping 3 ; Wang, Junmin 4 ; Xu, Ju 1 ;

作者机构： 1.Zhejiang Acad Agr Sci, Inst Qual & Stand Agroprod, State Key Lab Breeding Base Zhejiang Sustainable, Hangzhou, Peoples R China

2.Chinese Acad Agr Sci, Key Lab Biol & Genet Improvement Oil Crops, Minist Agr, Oil Crops Res Inst, Wuhan, Peoples R China

3.Zhejiang Univ, Coll Biosyst Engn & Food Sci, Hangzhou, Peoples R China

4.Zhejiang Acad Agr Sci, Inst Crops & Nucl Technol Utilizat, Hangzhou, Peoples R China

关键词： gene editing; CRISPR; Cas; accurate detection; dPCR; quantity analysis

期刊名称：FRONTIERS IN PLANT SCIENCE （ 影响因子：5.753； 五年影响因子：6.612 ）

ISSN： 1664-462X

年卷期： 2020 年 11 卷

页码：

收录情况： SCI

摘要： Gene-editing techniques are becoming powerful tools for modifying target genes in organisms. Although several methods have been reported that detect mutations at targeted loci induced by the CRISPR/Cas system in different organisms, they are semiquantitative and have difficulty in the detection of mutants in processed food samples containing low initial concentrations of DNA and may not accurately quantify editing frequency, especially at very low frequencies in a complex polyploid plant genome. In this study, we developed a duplexed dPCR-based method for the detection and evaluation of gene-editing frequencies in plants. We described the design, performance, accurate quantification, and comparison with other detection systems. The results show that the dPCR-based method is sensitive to different kinds of gene-editing mutations induced by gene-editing. Moreover, the method is applicable to polyploid plants and processed food samples containing low initial concentrations of DNA. Compared with qPCR and NGS-based methods, the dPCR method has a lower limit of detection (LOD) of the editing frequency and a better relationship with the expected editing frequency in detecting the edited region of gene-edited rice samples. Taken together, the duplexed dPCR assay is accurate and precise, and it will be a powerful tool for the detection and evaluation of gene-editing frequencies in plants in gene-editing technology.

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