Target-triggered CRISPR–Cas13a autocatalysis-driven amplification strategy for one-step detection of circadian clock gene†

Abstract

The precise and sensitive detection of circadian clock genes is crucial for understanding their roles in regulating biological processes and their implications in various diseases. Here, we present a CRISPR/Cas13a-powered autocatalytic cleavage circuit (CRISPR-ACC) for one-step detection of the BMAL1 mRNA, a key component of the circadian clock. The CRISPR-ACC system utilizes a three-stranded RNA probe for Cas13a-mediated collateral cleavage and autocatalysis-driven amplification, resulting in attomolar sensitivity and single-base mismatch discrimination. We demonstrate the efficacy of CRISPR-ACC in detecting BMAL1 from cell extracts, with results that strongly correlate with RT-PCR. Our method provides a simple, rapid, and highly specific approach for evaluating circadian clock genes, with potential applications in circadian-related disease diagnosis. The CRISPR-ACC system can be readily adapted to detect other clock genes, highlighting its versatility and promise for advancing our understanding of circadian rhythms.