Abstract 3794: Sensitive detection of microRNA biomarkers of pancreatic cancer using one-pot endonucleolytically exponentiated rolling circle amplification by CRISPR-Cas12a
Authors: Yan He , Han Song , Hughes Steven , Zeng Yong 
Published: April 2023

Abstract
While miRNAs offer a promising source for developing diagnostic cancer biomarkers, the progress towards clinical utilities remains largely limited, due in part to the long-standing challenge in sensitive, specific, and robust detection of miRNAs in human biofluids. Emerging next-generation molecular technologies, such as the CRISPR-based methods, promise to transform nucleic acid testing. The prevailing strategy used in existing CRISPR-based methods is to orthogonally hyphenate two separate reactions for pre-amplification, e.g., rolling circle amplification (RCA), and amplicon detection by Cas12a/13a trans-cleavage in tandem. Thus, existing CRISPR-based miRNA assays require multiple manual steps and lack the analytical performance of the gold standard, RT-qPCR. Radically deviating from the existing strategies, we developed a one-step, one-pot isothermal miRNA assay termed “Endonucleolytically eXponenTiated Rolling circle Amplification with the dual-functional CRISPR-Cas12a” (EXTRA-CRISPR) for miRNA detection with RT-qPCR-like analytical performance, including high sensitivity with a single-digit fM detection limit, single-nucleotide specificity, and rapid and flexible turnaround (from 20 min to 3 h for the entire analysis depending on targets and samples). We adapted the EXTRA-CRISPR assay to quantifying miRNA biomarkers in extracellular vesicles (EVs), a major carrier of miRNAs in human biofluids, for liquid biopsy diagnosis of pancreatic ductal adenocarcinoma (PDAC). Using the EXTRA-CRISPR, we demonstrated highly sensitive and specific profiling of a panel of four miRNA markers (miR-21, miR-196a, miR-451a, and miR-1246) in plasma EVs (PDAC patients (n = 20) and healthy donors (n = 15)). Based on the individual EV-miRNA tests (AUC values ranging from 0.677 for miR-196a to 0.793 for miR-451a), an EV signature (EV-Sig) combining miR-21, miR-451a and miR-1246 was devised with a machine learning method to improve the diagnostic performance for PDAC (AUC = 0.853). The diagnostic performance of the EXTRA-CRSPR tests were rigorously validated by parallel RT-qPCR analysis of the same clinical samples, and the RT-qPCR signature combining the three markers confers a similar AUC of 0.874 for PDAC detection. Overall, the analytical and diagnostic performance of our method were shown to be comparable with that of the commercial RT-qPCR assays, while greatly simplifying the analysis workflow. Therefore, we envision that our technology provides a promising tool to advance miRNA analysis and clinical marker development for liquid biopsy-based cancer diagnosis and prognosis. Moreover, this simple and robust assay permitted direct coupling with a 3D printed smartphone-based portable device and the lateral flow assay for signal readout, demonstrating its potential adaptability to low-cost point-of-care diagnostics.
Citation Format: He Yan, Song Han, Steven Hughes, Yong Zeng. Sensitive detection of microRNA biomarkers of pancreatic cancer using one-pot endonucleolytically exponentiated rolling circle amplification by CRISPR-Cas12a. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3794.