A visual CRISPR/dCas9-mediated enzyme-linked immunosorbent assay for nucleic acid detection with single-base specificity

Abstract

Specific and economical nucleic acid detection is crucial for molecular diagnoses in resource-limited settings. Various facile readout approaches have been developed for nucleic acid detection, but they have limited specificity. Herein, nuclease-dead Cas9 (dCas9)/sgRNA was used as an excellent DNA recognition probe system to develop a visual clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9-mediated enzyme-linked immunosorbent assay (ELISA) for specific and sensitive detection of cauliflwer mosaic virus 35s (CaMV35S) promoter in genetically modified (GM) crops. In this work, the CaMV35S promoter was amplified with biotinylated primers, and then precisely bound with dCas9 in the presence of sgRNA. The formed complex was captured by antibody-coated microplate and bound to a streptavidin-labeled horseradish peroxidase probe for the visual detection. Under the optimal conditions, dCas9-ELISA could detect CaMV35s promoter as low as 12.5 copies μL−1. Moreover, the proposed method was capable to distinguish the target sequence with single-base specificity. Coupled with one-step extraction and recombinase polymerase amplification, dCas9-ELISA can identify actual GM rice seeds within 1.5 h from sampling to results without expensive equipment and technical expertise. Therefore, the proposed method offers a specific, sensitive, rapid and cost-effective detection platform for molecular diagnoses.