Development of A NovelBlood-MicroRNA Handy Detection System with CRISPR
MicroRNAs (miRNA), serve as critical gene expression regulators at the transcriptional and post-transcriptional levels, have also been found as important blood-based biomarkers for early detection of cancers. However, their current in vitro detection methods are relatively complex, costly and low sensitive. Our project attempts to establish a novel in vitro miRNA detection system which is efficient, sensitive, specific, low-cost and output-visible. In this system, CRISPR-Cas9 technique is modified to integrate with split-HRP systems. The advanced chain-replacement and rolling circle amplification technologies are also involved and optimized.
To proof our concept and demonstrate our designs, miR let-7a, a typical lung cancer biomarker in serum, was selected as a detection prototype. As expected, gradient dilution miR let-7a showed significant signal variation both resolved in water and serum. The results were exceedingly in consistent with the modeling data, which obtained parameters from experiment results and theoretical calculation and in turn provided important experimental conditions for wet-lab. Finally, our approach was resorted to the clinical amplification by comparing the serum miR let-7a concentrations between non-small cell lung cancer (NSCLC) and healthy volunteers. Results confirmed the significant miR let-7a down-regulation in NSCLC, and demonstrated the clinical practicability of our detection system.
Not only for the analyses of miRNA, this system may ideally be compatible for the detection of various series of small non-coding RNAs. To our knowledge, we are the first to use the CRISPR-Cas9 system as a small non-coding RNA monitor in vitro. Its establishment and further development might provide a new approach for rapid and low-cost cancer screening, virus detection and curative efficacy assessment.