Sensitive detection Cu/Zn SOD mRNA with CRISPR/Cas9

Abstract

Cu/Zn SOD mRNA plays a critical role in protecting cells from oxygen toxicity by regulating the expression of the Cu/Zn SOD enzyme. In this work, we developed a biosensor, termed the Cas9-sgRNA/blocker system, for the analysis of Cu/Zn SOD mRNA. The biosensor was assembled by hybridizing a crRNA with a complementary blocker strand, which was then complexed with the Cas9 nuclease. In the presence of the target mRNA, competitive binding between the blocker strand and the mRNA restored Cas9-sgRNA cleavage activity, enabling enzymatic cleavage of adjacent fluorescent dsDNA reporters. Fluorescence measurements were performed at λex/λem = 488/525 nm, yielding a detection limit of 1.40 nmol L⁻¹. This method demonstrated excellent selectivity for Cu/Zn SOD mRNA, as evidenced by its successful application to the detection of Cu/Zn SOD mRNA in real samples.