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

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 T 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 transgenic plants, which will be widely used in the area of plant gene editing.

Application of the CRISPR/Cas9 system for modification of flower color in Torenia fournieri

“…Several methods currently exist to assess the outcome of genome editing, such as a PCR/restriction enzyme assay, high-resolution melting (HRM) analysis, and T7 Endonuclease I and TaqMan qPCR assays [ 32 ]. Although these methods are useful for screening mutations introduced by the CRISPR/Cas9 system, sequencing analysis provides the most direct evidence to confirm genome editing.…”

Improving CRISPR‐Cas‐mediated RNA targeting and gene editing using SPLCV replicon‐based expression vectors in Nicotiana benthamiana

“…After 6 days of infiltration, a higher accumulation of nucleases, crRNA transcripts and the nuclease protein was found in SV‐infiltrated leaves than in TV‐infiltrated ones (Figures 1r and s). The average relative amplification values of the target sites obtained using the qPCR‐based method were approximately 1.01 and 0.90 in NT‐infiltrated and TV‐infiltrated samples, respectively (Figures 1t and u; Peng et al , 2018). Such a notion suggests that the mutation frequency for TVs was approximately 10%, and this result was similar to a previous report (Bernabé‐Orts et al , 2019).…”

Genomic interventions for sustainable agriculture

“…It quickly became evident that producing gene‐edited plants became more straightforward than detecting edited plants, particularly when the altered phenotype was not evident visually. Various groups have developed rapid phenotyping tests to more efficiently screen plants for the most desirable edit(s) (Peng et al ., 2018).…”