CRISPR/dCas9-Assisted On-Bead Multiplex Detection (BeadPlex2) for Genetically Modified Crops

This study leverages the precise recognition ability of CRISPR/dCas9 and the Raman coding feature of the gap-enhanced Raman tag-encoded magnetic beads (MagGERTs) to create a unique on-bead nucleic acid detection platform (BeadPlex2) for accurate and multiplex nucleic acid detection, which was proven to be applicable for the identification of diverse genetically modified (GM) events. Five distinct MagGERTs (MB@Au^Ra) encoded with different Raman reporters (Ras) were constructed, followed by the conjugation of dCas9/single guide RNA (sgRNA) complexes in which the sgRNAs were explicitly designed for different target genes of GM events. These coding units could recognize and capture target double-stranded nucleic acid (dsDNA) sequences by the dCas9/sgRNA complexes. Then, SYBR Green I was applied to highlight positive beads by binding to target dsDNA due to its fluorescent emission under an imaging system. Decoding Raman signals from the Ras of the MagGERTs achieved the high-specific identification of GM events. Our BeadPlex2 platform has been demonstrated to be applicable for detecting GM maize and soybean seeds with high accuracy comparable to qPCR. This platform opens a new way to detect multiple target nucleic acids simultaneously and offers a powerful strategy for identifying genetically modified organisms.