Development of highly sensitive one-pot ERA-CRISPR assays for on-site detection of CaMV35S promoter and NOS terminator in genetically modified crops

Abstract

The integration of nucleic acid isothermal amplification with CRISPR/Cas12a detection technologies has significantly advanced molecular diagnostics. However, the challenge of integrating both assays into a one-pot reaction to reduce operational complexity and the risk of aerosol contamination has persisted. In this study, we developed a one-pot assay that combines these two powerful tools to improve the detection of genetically modified organisms (GMOs). Targeting the widely used Cauliflower Mosaic Virus 35S promoter (P-CaMV 35S) and the nopaline synthase terminator (T-NOS) from Agrobacterium tumefaciens in genetically modified (GM) crops, we employed a definitive screening design (DSD) approach to optimize the balance between ERA amplification and Cas12a activity. This optimization was achieved by enhancing ERA amplification and precisely adjusting the concentrations of Cas12a and other reaction components, resulting in an efficient and streamlined process. The optimized one-pot ERA-CRISPR/Cas12a system achieved a detection sensitivity of 10 copies per reaction for both P-CaMV 35S and T-NOS within 40 min at 40 °C, and was capable of detecting GMO content as low as 0.1% in spiked samples. Moreover, with minimal equipment requirements, such as an LED blue light and a smartphone for result interpretation, this method is highly user-friendly. Combining rapid detection, high specificity, and operational simplicity, this system represents a significant advancement in supporting GMO regulation and global trade, and serves as a promising model for the development of future nucleic acid-based assays.