A sensing strategy combining T7 promoter-contained DNA probe with CRISPR/Cas13a for detection of bacteria and human methyltransferase

Abstract

Abnormal DNA methylation is closely related to the occurrence and development of many diseases. The determination of human DNA methyltransferase activity and the screening of its inhibitors are extreme important for the diagnosis and the treatment of methylation-related diseases in clinic. Most of the current detection methods have the disadvantages of sophisticated design, high cost and low detection limit. By combining T7 promoter-contained DNA probe as the substrate for methyltransferase with CRISPR/Cas13a sensing strategy, a novel fluorescent sensing platform is designed to achieve simple, specific, sensitive detection of bacteria DNA methyltransferase (DNA-(N-6-adenine)-methyltransferase, Dam MTase) and also human methyltransferase (DNA (cytosine-5)-methyltransferase 1, Dnmt1). A hairpin DNA probe designed for Dam MTase and a double strand DNA probe for Dnmt1 are both methylated followed by the methylation-dependent site-specific cleavage, which result a T7 promoter-contained product and a T7 promoter-free one to respectively open and close the transcription and subsequent CRISPR/Cas13a target-initiated cleavage of fluorescence-labeled reporter RNA. In virtue of the specificity of methylation-dependent cleavage of probe, the efficient transcription amplification and CRISPR/Cas13a sequence-specific sensing, this strategy exhibited remarkable specificity and sensitivity, with the limit of detection of 3.10 × 10−5 U/mL for Dam MTase. Moreover, Dnmt1 activity in MCF-7 cells was detected and the inhibition of Apt. #9 was evaluated. This strategy for methyltransferase detection is convenient and efficient for inhibitor discovery and early cancer diagnosis.