Universal CRISPR/Cas12a-associated aptasensor suitable for rapid detection of small proteins with a plate reader.

With the discovery of the collateral cleavage activity, CRISPR/Cas12a has recently been identified as a key enabling approach in novel DNA biosensor development. Despite its remarkable success in nucleic acid detection, realizing a universal CRISPR/Cas biosensing system for non-nucleic acid targets remains challenging, particularly at extremely high sensitivity ranges for analyte concentrations lower than the pM level. DNA aptamers can be designed to bind to a range of specific target molecules, such as proteins, small molecules, and cells, with high affinity and specificity through configuration changes. Here, by harnessing its diverse analyte-binding ability and also redirecting the specific DNA-cutting activity of Cas12a to selected aptamers, a simple, sensitive, and universal biosensing platform has been established, termed CRISPR/Cas and aptamer-mediated extra-sensitive assay (CAMERA). With simple modifications to the aptamer and guiding RNA of Cas12a RNP, CAMERA demonstrated 100 fM sensitivity for targeting small proteins, such as IFN-γ and insulin, with less than 1.5-h detection time. Compared with the gold-standard ELISA, CAMERA achieved higher sensitivity and a shorter detection time while retaining ELISA's simple setup. By replacing the antibody with an aptamer, CAMERA also achieved improved thermal stability, allowing to eliminate the requirement for cold storage. CAMERA shows potential to be used as a replacement for conventional ELISA for a variety of diagnostics but with no significant changes for the experimental setup.