MicroRNA-triggered dCas9 beacon for cancer detection

Abstract

Cancer is the second leading cause of death in the United States. Early detection has a major influence on receiving effective treatment and increasing the chance of survival. One such target for detection of cancer is microRNA. Deregulation of microRNA have been linked to a variety of cancers; which combined with their small size of 22 nucleotides makes them a popular target for cancer detection. ☐ In this work, an in vitro method for sensitive, rapid detection of cancer-relevant microRNA has been developed. The key of the assay relies on the ability of the type II catalytically inactive Cas9 protein, when complexed with its single-guide RNA, to recognize and bind to any designed double-stranded DNA target. The presence of a cancer-relevant microRNA triggers a series of toehold displacement reactions resulting in a fully assembled molecular beacon labeled by a fluorophore-quencher pair. Upon assembly of the beacon, a dCas9-sgRNA complex binds and displaces a quencher strand for fluorescence signal. This dCas9 beacon provides a microRNA detection limit at nanomolar concentrations.