Branched Split Activator-Regulated Cas12a Biosensor For Methyltransferases Detection

DNA methyltransferases (MTases) are important epigenetic enzymes involved in DNA methylation regulation and have attracted considerable interest as analytical targets in disease-related epigenetic studies. However, traditional detection methods are constrained by laboratory-bound, sophisticated instrumentation and time-consuming protocols, while emerging approaches either depend on complex enzymatic amplifications or suffer from uncontrolled background signals, limiting their bioanalysis utility for precision-medicine-related applications. Inspired by the structure-controlled activation mechanism of CRISPR/Cas12a, we designed a branched split structure to regulate Cas12a for stepwise enzymatic activation and established the Cas12a-induced DNA MTase Sensor (CDMS), which enables highly sensitive MTase activity detection without the need for nucleic-acid amplification step. The core strategy lies in designing split-and-reconstituted Cas12a-activating DNA fragments with steric hindrance effects. This design significantly inhibits nonspecific Cas12a activation while efficiently restoring its activity upon removal of steric hindrance in response to DNA MTase activity. This method achieves ultra-low detection limit of 1.06 × 10-6 U/mL for M.SssI MTase and 7.41 × 10-8 U/mL for Dam MTase, exhibiting excellent specificity and robust applicability for MTase inhibitor screening. We also developed a palm-sized portable optical device, named iCas-Analyzer, to further explore the CDMS's potential for point-of-care testing (POCT), which can be easily operated by nonexpert end users. With device integration, the CDMS extends CRISPR-based diagnostics into the field of epigenetics, holding its potential for future decentralized MTase analysis and POCT-oriented assay development.