CRISPR/dCas9-assisted SERS sensing for transgenic crops based on two-factor verification of 35S promoter and Nos terminator

A nucleic acid analysis platform was built based on the combination of the recombinase polymerase amplification (RPA)-aided CRISPR/dCas9 and surface-enhanced Raman scattering (SERS) technologies, which enables the simultaneous detection of dual nucleic acid fragments, for the purpose of identifying transgenic crops . Two SERS-active nanoprobes (AgNP@Ra@dCas9/sgRNA) were fabricated by grafting the single RNA (sgRNA)-activated dCas9 protein to silver nanoparticles that have been decorated with two different Raman reporters (Ra). RPA primers endow target genes with biotin end that can be captured by the streptavidin-modified magnetic bead (MB@SA), followed by the conjugation of SERS nanoprobes. Thus, strong SERS signals would be detected above the MBs. This SERS platform has been applied for identifying transgenic crops based on the sequence designs specifically for the 35S promoter and Nos terminator simultaneously. Double confirmation of target genes can ensure the accuracy of detection. The whole sensing process, including RPA, dCas9 activation, and target gene identification, can be completed in one tube, within 80 min. This CRISPR/dCas9-SERS analytical platform has been proved to be feasible in detecting plant samples, indicating one successful attempt of the CRISPR/dCas9-SERS systems in multi-target detection. • A combination of CRISPR/dCas9 and SERS technology for multi-target detection. • Highly sensitive SERS and amplification of RPA allow for ultra-low concentration DNA detection. • Magnetic bead enrichment for analytes is remarkable.