Identification of transgenic crops by CRISPR/Cas12a-assisted magnetic surface-enhanced resonance Raman spectroscopy

Rapid and reliable identification of genetically modified (GM) and non-GM crops is in urgent demand and remains a significant challenge in food safety. Here, a new and reliable Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-mediated magnetic surface-enhanced resonance Raman scattering (SERRS) biosensing is reported to identify specific gene sequences of transgenic crops (CaMV 35 S promoter and Nos terminator). This assay mainly combines the gene-specific recognition ability of CRISPR, the ultra-high sensitivity due to the Raman resonance effect, and the simple separation property of magnetic nanomaterials. A multifunctional SERRS-active nanoprobe was designed by decorating a gold-coated magnetic bead with the G-triplet deoxyribonucleic acid (DNA) sequences that enable a high loading of Raman reporters (methylene blue) inserted in G-triplet DNAs. The assay is achieved by the recognition of the CRISPR/Cas12a protein for bound target DNAs and the activation of the unlimited trans-cleavage action on the G-triplet DNAs, which causes a release of methylene blue above the nanoprobes and a reduction of the SERRS signal. Gel electrophoresis experiments imply that the designed crRNAs have good recognition and cleavage function for target DNAs and verify the feasibility of the experimental scheme. This SERRS assay exhibits a linear response of 4.15×10-12 M to 4.15×10-9 M with a pM detection level. Tests of the GM and non-GM crops of actual crop samples have been realized, which implies that this CRISPR-mediated magnetic SERRS method is available for assessing natural samples and working for GM crop regulation.