DNA polymerase mediated CRISPR/Cas12a trans-cleavage for dual-mode quantification of uracil DNA glycosylase activity

Since an unusual expression of uracil-DNA glycosylase (UDG) is often associated with the pathogenesis of numerous disorders, the detection of UDG activity is regarded as a promising application in disease diagnosis. Here, we develop a DNA polymerase-mediated CRISPR/Cas12a trans cleavage strategy, which can achieve dual-mode determination of UDG activity. By introducing a hairpin DNA probe containing a single uracil base, the probe undergoes specific cleavage and elongation under the existence of UDG only, thus activating the trans cleavage of ssDNA regardless of its length and sequence. To accommodate different detection modes, the ssDNA was further modified by fluorophore-quencher pairs or designed for connecting magnetic microparticles (MMPs) and polystyrene microparticles (PMPs). Finally, the UDG activity is quantified by fluorescence signal and microparticle accumulation length on a microfluidic chip visible to the naked eye. This strategy provides a detectable minimum UDG concentration of 0.00047 U/mL for fluorescent mode and 0.0048 U/mL for microfluidic mode. Furthermore, we performed the UDG inhibition test and detected UDG activity in cell lysates, suggesting its potential for inhibitor screening and detection of UDG in biological samples.