Dual detection of hypervirulent genes of <i>Klebsiella pneumoniae</i> using a single CRISPR-Cas12a system modulated using entropy-driven circuits

A set of different CRISPR-based analyses have been extensively used to detect nucleic acids owing to their characteristics of being sensitive, specific, rapid and easy to operate. However, one of its major challenges is application to multiplex detection (https://pubmed.ncbi.nlm.nih.gov/31460243/) using a single CRISPR-Cas system because the detection relies on the indiscriminate <i>trans</i>-cleavage activity of the Cas protein. Here we developed a CRISPR-based dual gene assay system for hypervirulent <i>Klebsiella pneumoniae</i> (hv<i>Kp</i>) by integrating two parallel entropy-driven circuits (EDCs) and the <i>trans</i>-cleavage activity of the Cas12a protein towards fluorescent substrate reporters. The EDC reaction released two different fluorescent reporters and the same trigger probe according to different targets. The trigger probe activated the <i>trans</i>-cleavage activity of Cas12a towards reporter probes to achieve the purpose of the dual detection of different genes with a single CRISPR-Cas12a system in one tube. Furthermore, the assay system was designed to detect different mRNA sequences (<i>rmpA</i> and <i>peg-344</i>) of hv<i>Kp</i>, requiring no reverse transcription procedure and providing technical support for the identification of hv<i>Kp</i> and classical <i>Klebsiella pneumoniae</i> (c<i>Kp</i>). The detection limits for <i>rmpA</i> and <i>peg-344</i> were as low as 0.10 fmol L^-1 and 0.17 fmol L^-1, respectively, within 25 minutes under isothermal conditions. It enabled rapid dual detection of hv<i>Kp</i> in a single tube using only a CRISPR-Cas12a system, demonstrating great potential for point-of-care testing due to its high sensitivity, low cost, and robust ability to detect various mRNA biomarkers.