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

The Plant Journal : for Cell and Molecular Biology
Cheng PengJunfeng Xu

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 sa...Continue Reading

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Citations

Apr 16, 2020·G3 : Genes - Genomes - Genetics·Renyu LiCristian H Danna
Sep 3, 2020·Plant Biotechnology Journal·Abhishek BohraRajeev Kumar Varshney
Dec 7, 2018·BMC Plant Biology·Masahiro NishiharaKeisuke Tasaki
Jan 6, 2021·International Journal of Molecular Sciences·Jaiana MalabarbaEmilie Vergne
May 13, 2021·Plant Cell Reports·Sukumar BiswasJianxin Shi

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