CRISPR/Cas12a technology is used for nucleic acid detection due to its specific recognition function and non-specific single-stranded DNA cleavage activity. Here, we developed a fluorescence visualisation detection method based on PCR and CRISPR/Cas12a approaches. The method was used to detect the nopaline synthase terminator (T-nos) of genetically modified (GM) crops, circumventing the need for expensive instruments and technicians. For enhanced sensitivity and stability of PCR-CRISPR/Cas12a detection, we separately optimised the reaction systems for PCR amplification and CRISPR/Cas12a detection. Eleven samples of soybean samples were assessed to determine the applicability of the PCR-CRISPR/Cas12a method. The method could specifically detect target gene levels as low as 60 copies in the reaction within 50 min. In addition, accurate detection of all 11 samples confirmed the applicability. The method is not limited by large-scale instruments, making it suitable for mass detection of transgenic components in plants in the field. In conclusion, we developed a new, accurate, rapid, and cost-effective method for GM detection.

Effective regulation of gene-edited products and resolution of public concerns are the prerequisites for the industrialization of gene-edited crops and their derived foods. CRISPR-associated protein, the core element of the CRISPR system, requires to be regulated. Thus, there is an urgent need to establish qualitative and quantitative detection methods for the Cas gene. In the present study, the primers and probes were designed and screened for Cas12a (Cpf1), which is the most commonly used target site in gene editing; we performed PCR system optimization, determined the optimal primer concentration and annealing temperature, and established qualitative PCR and quantitative PCR (qPCR) assays for detecting Cpf1 in gene editing by specificity and sensitivity tests. In specificity testing, qualitative PCR and qPCR methods could 100% detect samples containing Cpf1 DNA, while the detection rate of other samples without Cpf1 was 0%. In the assay sensitivity test, the limit of detection of qualitative PCR was 0.1% (approximately 44 copies), and the limit of detection of the qPCR method was 14 copies. In the stability test, both the qualitative PCR and qPCR methods were repeated 60 times at their corresponding lowest detection limit concentrations, and the results were positive. Thus, the qualitative and quantitative assays for Cpf1 are specific, sensitive, and stable. The method provides technical support for the effective monitoring of gene-edited products and their derived foods in the future.

The safety and unintended effects of genetically modified (GM) crops have been the focus of public attention. Transcriptome analysis is a powerful tool to assess the potential impact of genetic modification on plant genomes. In this study, three transgenic (KMD, KF6, and TT51-1) and three non-transgenic (XS11, MH86, and MH63) rice varieties were assessed at the genomic and protein levels. The results of polymerase chain reaction (PCR) and Cry1Ab/1Ac speed test strips showed that the Bt gene was successfully expressed in transgenic rice. The results of RNA-seq analysis to analyze the unintended effects of transgenic Bt rice showed fewer differentially expressed genes (DEGs) between the transgenic and non-transgenic rice varieties than among the different varieties. Meanwhile, the results of principal component analysis and cluster analysis found no significant genetic variation between the transgenic and non-transgenic rice varieties, except for the presence of Bt in transgenic rice. There were only two co-upregulated DEGs and no co-downregulated DEGs among three comparison groups. Although there were various DEGs among the groups, the two co-upregulated DEGs were not related to any significantly enriched gene ontology (GO) term or Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, indicating that the differences among the subgroups were more likely caused by complex environmental or genetic factors, rather than unintended effects due to Bt expression. This study provides useful information to further explore the unexpected effects and safety of GM rice.