Droplets Cas13a-RPA measurement delineates potential role for plasma circWDR37 in colorectal cancer

Abstract

Colorectal cancer (CRC) is one of the most prevalent forms of cancer. CircRNAs have emerged as promising biomarkers for cancer diagnosis and prognosis evaluation. However, novel circRNAs as potential biomarkers for CRC still need further exploration and validation, and precise detection methods are yet to be developed. Herein, we report for the first time the use of droplets Cas13a to detect the circWDR37 as a biomarker of CRC. The arraystar circRNA microarray assays, functional experiments in vitro and in vivo, and qPCR were performed to discover and validate that circWDR37 is a biomarker for early screening and prognosis evaluation of CRC. A new technology named µDCR, which accurately detects circWDR37, has been developed by combining microfluidic droplets with CRISPR/Cas13a and recombinase polymerase amplification (RPA). Meanwhile, the role of crowding agent in improving the performance of Cas13a was uncovered. The 4% polyethylene glycol 8000 and 3% dextran-10 significantly improved the response speed and sensitivity of one-pot Cas13a-RPA reaction. The detection limit of circWDR37 by µDCR was found to be 10 copies/mL, which is higher than that of qPCR. The clinical sample findings demonstrated that circWDR37 detection can be utilized to effectively screen for CRC at an early stage and enable accurate assessment of prognosis. CircWDR37 is confirmed as a groundbreaking biomarker for both diagnosis and prognosis evaluation in CRC patients. Furthermore, our innovative µDCR method for detecting circWDR37 demonstrates impressive attributes such as streamlined operation, rapidity, and high-throughput, making it an optimal technology platform for the noninvasive screening of CRC.

Keywords

circRNA / colorectal cancer / CRISPR/Cas13a / crowding agent / droplet

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2024 The Author(s). Aggregate published by SCUT, AIEI, and John Wiley & Sons Australia, Ltd.