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High-throughput detection and screening of plants modified by gene editing using quantitative real-time polymerase chain reaction

文献类型： 外文期刊

作者： Peng, Cheng 1 ; Wang, Hua 1 ; Xu, Xiaoli 1 ; Wang, Xiaofu 1 ; Chen, Xiaoyun 1 ; Wei, Wei 1 ; Lai, Yongmin 1 ; Liu, Guoqua 1 ;

作者机构： 1.Zhejiang Acad Agr Sci, Inst Qual & Stand Agroprod, State Key Lab Breeding Base Zhejiang Sustainable, Hangzhou 310021, Zhejiang, Peoples R China

2.Univ Queensland, Sch Agr & Food Sci, St Lucia, Qld, Australia

3.Anhui Sci & Technol Univ, Coll Agr, Fengyang, Peoples R China

4.Jilin Acad Agr Sci, Agrobiotechnol Res Inst, Changchun 130033, Jilin, Peoples R China

关键词： gene editing; clustered regularly interspaced short palindromic repeats nucleases; detection; quantitative PCR; high-throughput; high-throughput

期刊名称：PLANT JOURNAL （ 影响因子：6.417； 五年影响因子：7.627 ）

ISSN： 0960-7412

年卷期： 2018 年 95 卷 3 期

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 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 T-0 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 T-0 transgenic plants, which will be widely used in the area of plant gene editing. Significance Statement An accurate and high-throughput method is presented that uses qPCR for the detection and screening of plants modified by gene editing. This technology simplifies and facilitates the detection of induced mutations and is especially useful for evaluating the efficiency of gene editing and screening of gene-edited plants from many candidates and is a significant improvement of existing methods.

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