Integrated CRISPR/Cas12a with terminal protection strategy for homogeneous protein assay

Developing appropriate protein assay methods is necessary, as protein assays play an important role in molecular diagnosis, drug screening, and biomedical research. This article presents a universal homogeneous method based on the CRISPR/Cas12a system and the terminal protection strategy for protein assays. Initially, a single-stranded DNA (ssDNA) labeled with a small molecule at its 3'-end was designed to respond to the target protein and serve as an activator of Cas12a. In the absence of the target protein, small-molecule-linked ssDNA was effectively cleaved using exonuclease I (Exo I). The lack of the activator resulted in the trans-cleavage inactivity of Cas12a, leading to low fluorescence in the detection solution. However, in the presence of the target protein, the specific binding of the target protein to the small molecules on the activator protected the activator against digestion by Exo I. The remaining intact activator triggered the trans-cleavage activation of Cas12a, producing strong fluorescence. Based on this principle, our method achieved fluorescence enhanced detection of the target protein. After experimental condition optimization, our method achieved highly sensitive and specific protein detection in two models: folate receptor and streptavidin. The excellent performance of the fluorescence method highlights the applicability and effectiveness of the CRISPR/Cas12a system in protein assays, and broadening its application scope.