An ultra-sensitive and specific UCBiosensor via CRISPR-Cas12a and UDG-mediated polymerase chain reaction

Abstract

DNA damage is an important cause of several diseases. Uracil DNA glycosylase (UDG) is a key enzyme in DNA damage repair and used as an early diagnostic marker and a prognostic indicator for cancer. Therefore, there is an urgent need to develop a simple, low-background, ultra-sensitive UDG activity detection method for clinical applications. At present, biosensors based on CRISPR-Cas12a have become a powerful diagnostic tool owing to their high sensitivity. Here, we have established an ultra-sensitive biosensor for detecting UDG activity based on CRISPR-Cas12a coupled UDG-mediated polymerase chain reaction (U-PCR), which has the advantages of simplicity, low background, ultra-sensitivity, and specificity. We used this biosensor to multiply amplify the UDG signal, and the detection limit reached 3.5 × 10−6 U/mL, which is one of the most sensitive methods for UDG activity detection. Finally, we detected UDG activity in the lysates of cancer cells, indicating that this method could be used to detect UDG activity in real samples. The UDG inhibition test showed that the method we established is a useful tool for screening UDG inhibitors. We believe that the UDG activity detection biosensor established in this study has potential applications in biomedical research and clinical diagnosis.