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)

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 pointof-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 1.48x10(-4) U/mL, 92.5 cells/mL, and 2.52x10(-4) U/mL, respectively. Besides, we achieved naked eye and timely detection of T4 PNK, telomerase, or APE1 activity low to 2.5x10(-3) U/mL, 9.25x10(2) cells/mL, and 2.5x10(-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.Y

Automated summary

Enzymes are essential components of organisms, ensuring normal life activities. Abnormal enzyme activity can lead to dysfunction and disease. Establishing simple, sensitive, and timely detection of enzyme activity is crucial for biomedical testing and disease diagnosis and treatment. We developed a strategy for detecting T4 PNK, telomerase, or APE1 activity using enzyme extends activators to unlock CRISPR/Cas12a's ssDNase activity (EdU-CRISPR/Cas12a), achieving low detection limits through fluorescence and lateral flow assay methods. Our approach has the potential to greatly improve enzyme activity detection.