An ultrasensitive and point-of-care strategy for enzymes activity detection based on enzyme extends activators to unlock the ssDNase activity of CRISPR/Cas12a (EdU-CRISPR/Cas12a)

Abstract

• Proposing a concept of EdU-CRISPR/Cas12a firstly. • Applying the CRISPR/Cas12a into the detection of enzyme activity. • Achieving the ultrasensitive detection of T4 PNK, telomerase, or APE1 activity detection based on fluorescence assay. • Achieving the visible detection of T4 PNK, telomerase, or APE1 activity based on CRISPR-Cas12a-based lateral flow assay. As the indispensable parts of the beings, enzyme plays an essential role in guaranteeing the normal life activities of the organism. But abnormal enzyme activity may lead to the body dysfunction and even disease. Thus, establishing the simple, ultrasensitive, and timely detection of enzyme activity is of great significance to biomedical testing and diagnosis and treatment of disease. Here, we created a simple, ultrasensitive, and point-of-care strategy for T4 PNK, telomerase, or APE1 activity detection based on enzyme extends activators to unlock the ssDNase activity of CRISPR/Cas12a (EdU-CRISPR/Cas12a). We make full use of the enzymes’ activity, so that the primers are continuously extended to generate multiple activation regions which can initiate the ssDNase activity of the CRISPR/Cas12a. Based on fluorescence strategy, we achieved simple and ultrasensitive determination of T4 PNK, telomerase, or APE1 activity low to 1.48×10 −4 U/mL, 92.5 cells/mL, and 2.52×10 −4 U/mL, respectively. Besides, we achieved naked eye and timely detection of T4 PNK, telomerase, or APE1 activity low to 2.5×10 -3 U/mL, 9.25×10 2 cells/mL, and 2.5×10 -2 U/mL with the CRISPR-Cas12a-based lateral flow assay. We think the proposed EdU-CRISPR/Cas12a will play a meaningful role in establishing simple, ultrasensitive, and visual enzyme activity detection.