Construction of a transcription-driven CRISPR RNA auto-generation-mediated CRISPR-Cas12a system for sensitive detection of endogenous repair glycosylase

Base excision repair (BER) plays an essential role in the repair of DNA damage and maintenance of genomic integrity. As a major BER enzyme, human alkyladenine DNA glycosylase (hAAG) acts on various alkylated bases, and its abnormal activity may lead to elevated risks of different cancers. Herein, we develop a transcriptiondriven CRISPR RNA (crRNAs) auto-generation-mediated CRISPR-Cas12a system for sensitive and simple detection of hAAG. In this strategy, a hairpin probe containing a deoxyinosine base/T (I/T) pairs and a destroyed T7 promoter sequence serves not only as a probe for hAAG recognition but also as a template for transcription, efficiently avoiding nonspecific transcription. Target hAAG induces the hairpin probe cleavage and subsequent unfolding. The unfolded hairpin probe can initiate the KF polymerase-mediated polymerization reaction and subsequent T7 polymerase-mediated transcription reaction for the production of substantial crRNAs. The resultant crRNAs activate the trans-cleavage activity of CRISPR-Cas12a to cleave signal probes, generating an enhanced fluorescence signal. This strategy can measure hAAG with a limit of detection of 9.25 x 10-11 U/mu L and a broad linear range of 1 x 10- 10-1 x 10-2 U/mu L, and it may accurately measure endogenous hAAG at the single-cell level. Furthermore, the assay can be applied for inhibitor screening, kinetic analysis, and the differentiation of tumor cells from healthy cells, with great potential in disease diagnosis.