High‐throughput detection and screening of plants modified by gene editing using quantitative real‐time polymerase chain reaction |
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| Authors: | Cheng Peng Hua Wang Xiaoli Xu Xiaofu Wang Xiaoyun Chen Wei Wei Yongmin Lai Guoquan Liu Ian Douglas Godwin Jieqin Li Ling Zhang Junfeng Xu |
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| Affiliation: | 1. State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control, Institute of Quality and Standard for Agro‐Products, Zhejiang Academy of Agricultural Sciences, Hangzhou, China;2. School of Agriculture and Food Sciences, The University of Queensland, St Lucia, QLD, Australia;3. College of Agriculture, Anhui Science and Technology University, Fengyang, China;4. Agro‐Biotechnology Research Institute, Jilin Academy of Agricultural Sciences, Changchun, Jilin, China |
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| Abstract: | Gene editing techniques are becoming powerful tools for modifying target genes in organisms. Although several methods have been developed to detect gene‐edited organisms, these techniques are time and labour intensive. Meanwhile, few studies have investigated high‐throughput detection and screening strategies for plants modified by gene editing. In this study, we developed a simple, sensitive and high‐throughput quantitative real‐time (qPCR)‐based method. The qPCR‐based method exploits two differently labelled probes that are placed within one amplicon at the gene editing target site to simultaneously detect the wild‐type and a gene‐edited mutant. We showed that the qPCR‐based method can accurately distinguish CRISPR/Cas9‐induced mutants from the wild‐type in several different plant species, such as Oryza sativa, Arabidopsis thaliana, Sorghum bicolor, and Zea mays. Moreover, the method can subsequently determine the mutation type by direct sequencing of the qPCR products of mutations due to gene editing. The qPCR‐based method is also sufficiently sensitive to distinguish between heterozygous and homozygous mutations in T0 transgenic plants. In a 384‐well plate format, the method enabled the simultaneous analysis of up to 128 samples in three replicates without handling the post‐polymerase chain reaction (PCR) products. Thus, we propose that our method is an ideal choice for screening plants modified by gene editing from many candidates in T0 transgenic plants, which will be widely used in the area of plant gene editing. |
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| Keywords: | gene editing clustered regularly interspaced short palindromic repeats nucleases detection quantitative PCR high‐throughput high‐throughput |
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