Paired dCas9 design as a nucleic acid detection platform for pathogenic strains

Abstract

The wide application of molecular beacon probes in specific DNA detection, especially in the fast prototyping of pathogen DNA detection kits in point-of-care diagnostics, has been hindered by the nonflexible choice of target sequences and the unstable fluorophore output. We developed an in vitro DNA detection system consisting of a pair of dCas9 proteins linked to split halves of luciferase, named the Paired dCas9 (PC) reporter. Co-localization of the reporter pair to a ~46 bp target sequence defined by two single guide RNAs (sgRNAs) activated luciferase which subsequently generated highly intensified luminescent signals. Combined with an array design and statistical analyses, the PC reporter system could be programmed to access sequence information across the entire genome of the pathogenic Mycobacterium tuberculosis H37Rv strain. These findings suggest great potential for the PC reporter in effective and affordable in vitro nucleic acid detection technologies. In this article we highlighted the systems design from our previous researchworkon the PC reporter (Zhang et al, 2015)with a focuson methodology.

Keywords: Biosensor; Molecular Diagnostics; Nucleic acid detection; dCas9.