CRISPR/Cas12a collateral cleavage activity for simple and rapid detection of protein/small molecule interaction

• Published in: Biosensors & bioelectronics 2021-12, Vol.194, p.113587-113587, Article 113587
• Main Authors: Kim, Hansol, Lee, Seoyoung, Yoon, Junhyeok, Song, Jayeon, Park, Hyun Gyu
• Format: Article
• Language: English
• Subjects: Collateral cleavage; CRISPR/Cas; Fluorescent biosensor; Protein/small molecule interaction
• ISSN: 0956-5663; 1873-4235
• DOI: 10.1016/j.bios.2021.113587

To realize the full potential of the CRISPR/Cas system and expand its applicability up to the detection of molecular interactions, we herein describe a novel method to identify protein/small molecule interactions by utilizing the CRISPR/Cas12a collateral cleavage activity. This technique employs a single-stranded activator DNA modified with a specific small molecule, which would switch on the CRISPR/Cas12a collateral cleavage activity upon binding to crRNA within the CRISPR/Cas12a system. When the target protein binds to the small molecule on the activator DNA, the bound protein sterically hinders the access of the activator DNA to crRNA, thereby promoting less collateral cleavage activity of CRISPR/Cas12a. As a consequence, fewer reporter probes nearby are cleaved to produce accordingly reduced fluorescence signals in response to target protein. Based on this unique design principle, the two model protein/small molecule interactions, streptavidin/biotin and anti-digoxigenin/digoxigenin, were successfully determined down to 0.03 nM and 0.09 nM, respectively, with a fast and simple detection workflow (11 min). The practical applicability of this method was also verified by reliably detecting target streptavidin spiked in heterogeneous human serum. This work would provide great insight to construct novel strategies to identify protein/small molecule interaction by making the most of the CRISPR/Cas12a system beyond its superior capabilities in genome editing and molecular diagnostics. •A novel strategy is developed to identify protein/small molecule interactions by utilizing the CRISPR/Cas12a.•The developed strategy could detect streptavidin down to 0.03 nM.•The practical applicability of this strategy was verified by reliably detecting the streptavidin present in human serum.•This work would provide great insight for the construction of novel assay systems by utilizing CRISPR/Cas12a.