A CRISPR/Cas12a-Mediated Sensitive DNA Detection System for Gene-Edited Rice.

BACKGROUND: In recent years, genome editing technology represented by clustered regularly interspaced short palindromic repeat/CRISPR-associated nuclease 9 （CRISPR/Cas9） has been developed and applied in transgenic research and development, and transgenic products have been developed for a variety of applications. Gene editing products, unlike traditional genetically modified crops, which are generally obtained by target gene deletion, insertion, base mutation, etc., may not differ significantly at the gene level from conventional crops, which increases the complexity of testing. OBJECTIVE: We established a specific and sensitive CRISPR/Cas12a-mediated gene editing system to detect target fragments in a variety of transgenic rice lines and commercial rice-based processing products. METHODS: In this study, the CRISPR/Cas12a visible detection system was optimized for the visualization of nucleic acid detection in gene-edited rice. The fluorescence signals were detected by both gel electrophoresis and fluorescence-based methods. RESULTS: The detection limit of the CRISPR/Cas12a detection system established in this study was more precise, especially for low-concentration samples. In addition to achieving single-base detection in gene-edited rice, we showed that different base mutations in the target sequence have different detection efficiencies by sitewise variant compact analysis. The CRISPR/Cas12a system was verified via a common transgenic rice strain and commercial rice sources. The results proved that the detection method could not only be tested in samples with multiple mutation types but could also effectively detect target fragments in commercial rice products. CONCLUSION: We have developed a set of efficient detection methods with CRISPR/Cas12a for gene-edited rice detection to provide a new technical basis for rapid field detection of gene-edited rice. HIGHLIGHTS: The CRISPR/Cas12a-mediated visual detection method used to detect gene-edited rice was evaluated for its specificity, sensitivity, and robustness.

RNA Interference-Based Genetic Engineering Maize Resistant to Apolygus lucorum Does Not Manifest Unpredictable Unintended Effects Relative to Conventional Breeding: Short Interfering RNA, Transcriptome, and Metabolome Analysis.

The use of omics techniques to analyze the differences between genetic engineering organisms and their parents can identify unintended effects and explore whether such unintended effects will have negative consequences. In order to evaluate whether genetic engineering will cause changes in crops beyond the changes introduced by conventional plant breeding, we compared the extent of transcriptome and metabolome modification in the leaves of three lines developed by RNA interference (RNAi)-based genetic engineering and three lines developed by conventional breeding. The results showed that both types of plant breeding methods can manifest changes at the short interfering RNA (siRNA), transcriptomic, and metabolic levels. Relative expression analysis of potential off-target gene revealed that there was no broad gene decline in the three RNAi-based genetic engineering lines. We found that the number of DEGs and DAMs between RNAi-based genetic engineering lines and the parental line was less than that between conventional breeding lines. These unique DEGs and DAMs between RNAi-based genetic engineering lines and the parental lines were not enriched in detrimental metabolic pathways. The results suggest that RNAi-based genetic engineering do not cause unintended effects beyond those found in conventional breeding in maize.

Applicability of a General Analytical Approach for Detection of Genetically Modified Organisms: Collaborative Trial.

BACKGROUND: With the commercialization of genetically modified organisms (GMOs) in the market, laboratories have undergone a significantly increased workload. A universal analytical approach was designed to achieve cost-efficient and high-throughput GMOs screening with high specificity and accuracy. The approach provides accurate qualification of authorized and unauthorized GMOs. OBJECTIVE: This article describes the assessment of this analytical approach developed to detect the majority of commercialized GMOs over the world. METHOD: Seven elements and three events were detected by qPCR in a single laboratory to detect 59 commercialized GMOs. Certificated reference materials and food/feed samples from the Chinese market were also evaluated for the specificity, conformity, and robustness of this approach and were challenged in the interlaboratory study. RESULTS: The results showed that elements and events selected can best detect GMO presence with good specificity and sensitivity. The results showed a concordance between 97.5 and 99.56% and the variance between 0.65 and 12.88%, which is in line with the minimum requirement of analytical methods of GMO testing. CONCLUSIONS: The approach validated here can be used to manipulate GMO presence in food and feed and showed the capacity to manipulate GMO trace in the trade and domestic agriculture market in China. HIGHLIGHTS: A universal analytical approach used to track GMO presence was evaluated for its specificity, sensitivity, and robustness.