Premium
High‐throughput detection and screening of plants modified by gene editing using quantitative real‐time polymerase chain reaction
Author(s) -
Peng Cheng,
Wang Hua,
Xu Xiaoli,
Wang Xiaofu,
Chen Xiaoyun,
Wei Wei,
Lai Yongmin,
Liu Guoquan,
Godwin Ian Douglas,
Li Jieqin,
Zhang Ling,
Xu Junfeng
Publication year - 2018
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/tpj.13961
Subject(s) - amplicon , genome editing , biology , polymerase chain reaction , gene , crispr , mutant , computational biology , genetics , transgene , mutation
Summary 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.