Self-sustainable Catalytic Nucleic Acid Circuit Powered Hybrid CRISPR-Cas Systems for Point-of-Care Diagnosis of Circadian Clock Gene from Cell Lysates

The CRISPR-Cas13 systems have opened a new avenue for RNA detection due to their exceptional programmable collateral activity against ssRNA. However, most high-performance Cas13-based RNA sensors still suffer from some drawbacks because of the tradeoffs between readout device complexity and analytical sensitivity, which can limit their viability at the point-of-care (POC). To overcome these shortcomings, we herein report a novel target-responsive POC platform for translating RNA detection into a glucose test. Specifically, this platform combines the advantages of three techniques: a hybrid Cas13a/Cas12a system for both RNA recognition with single-base resolution and cascade enzymatic amplification, a self-sustainable catalytic nucleic acid circuit (SCC) with embedded uracil-base and poly-T bulges for Cas13a- and Cas12a-mediated collateral cleavage, respectively, and a portable glucose meter (PGM) for simple signal readout. The incorporation of an engineered ssDNA–invertase conjugate in SCC led to RNA-to-glucose signal transduction through the hydrolysis of sucrose to glucose. By targeting a 20-nt conserved region of BMAL1 mRNA (mBMAL1), a key circadian clock gene, we demonstrate that SCC can serve as an excellent CRISPR reporter, enabling the direct detection of unamplified mBMAL1 as low as 0.864 fM concurrently with good discrimination ability for single-base mismatch. More importantly, this integrated platform was successfully applied for POC diagnosis of mBMAL1 from different cell lysates, with results that strongly correlate with RT-PCR. Given its wide adaptability, we anticipate that such a CRISPR-SCC system can be easily modified to quantify other RNA biomarkers associated with circadian rhythm.