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

Area of Science:

• Molecular Biology
• Biotechnology
• Genetics

Background:

• Accurate detection of genetically modified (GM) events is crucial for food safety and regulatory compliance.
• Existing methods like qPCR can be time-consuming and may not be suitable for high-throughput multiplex detection.

Purpose of the Study:

• To develop a novel on-bead nucleic acid detection platform for accurate and multiplex identification of GM events.
• To leverage CRISPR/dCas9 and gap-enhanced Raman tag-encoded magnetic beads (MagGERTs) for enhanced specificity and sensitivity.

Main Methods:

• Constructed five distinct MagGERTs encoded with different Raman reporters (Ras).
• Conjugated dCas9/single guide RNA (sgRNA) complexes designed for specific GM target genes.
• Utilized SYBR Green I for fluorescent highlighting of captured double-stranded DNA (dsDNA).
• Decoded Raman signals from MagGERTs for GM event identification.

Main Results:

• The BeadPlex2 platform demonstrated high specificity in identifying diverse GM events.
• Accurate detection of GM maize and soybean seeds was achieved, comparable to qPCR performance.
• Successful simultaneous detection of multiple target nucleic acids was demonstrated.

Conclusions:

• The BeadPlex2 platform offers a powerful and accurate strategy for identifying genetically modified organisms (GMOs).
• This novel approach enables simultaneous detection of multiple nucleic acid targets, advancing GMO detection capabilities.