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A one-pot isothermal Cas12-based assay for the sensitive detection of microRNAs

Nature Biomedical Engineering volume 7, pages 1583–1601 (2023)Cite this article

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Abstract

The use of microRNAs as clinical cancer biomarkers is hindered by the absence of accurate, fast and inexpensive assays for their detection in biofluids. Here we report a one-step and one-pot isothermal assay that leverages rolling-circle amplification and the endonuclease Cas12a for the accurate detection of specific miRNAs. The assay exploits the cis-cleavage activity of Cas12a to enable exponential rolling-circle amplification of target sequences and its trans-cleavage activity for their detection and for signal amplification. In plasma from patients with pancreatic ductal adenocarcinoma, the assay detected the miRNAs miR-21, miR-196a, miR-451a and miR-1246 in extracellular vesicles at single-digit femtomolar concentrations with single-nucleotide specificity. The assay is rapid (sample-to-answer times ranged from 20 min to 3 h), does not require specialized instrumentation and is compatible with a smartphone-based fluorescence detection and with the lateral-flow format for visual readouts. Simple assays for the detection of miRNAs in blood may aid the development of miRNAs as biomarkers for the diagnosis and prognosis of cancers.

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Fig. 1: The one-pot EXTRA-CRISPR miRNA assay.
Fig. 2: Mechanistic studies of EXTRA-CRISPR.
Fig. 3: Comparison of the kinetics and detection sensitivity of RCA, one-pot EXTRA-CRISPR and multi-step CRISPR-assisted RCA assays.
Fig. 4: Optimization of the one-pot EXTRA-CRISPR miR-21 assay.
Fig. 5: Quantitative profiling of EV-derived miRNAs.
Fig. 6: One-pot miRNA analysis for the diagnosis of pancreatic cancer.
Fig. 7: Assessment of the EXTRA-CRISPR assay for low-cost POC testing.

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Data availability

The main data supporting the findings of this study are available within the paper and its Supplementary Information. The raw and analysed datasets generated during the study are available for research purposes from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank the University of Florida Clinical and Translational Science Institute Biorepository Facility for providing human plasma specimen. We thank Y. Li for helping with TEM analysis and G. Tushoski for helping with culturing cells and collecting conditioned medium. This study was supported in part by grants R01 CA243445, R33 CA214333, R33 CA252158A1 and R01 CA260132 from the National Institutes of Health.

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Authors and Affiliations

  1. Department of Chemistry, University of Florida, Gainesville, FL, USA

    He Yan, Yunjie Wen, Zimu Tian, Nathan Hart & Yong Zeng

  2. Department of Surgery, University of Florida College of Medicine, Gainesville, FL, USA

    Song Han & Steven J. Hughes

  3. J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, FL, USA

    Yong Zeng

  4. University of Florida Health Cancer Center, Gainesville, FL, USA

    Yong Zeng

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  1. He Yan
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  2. Yunjie Wen
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  3. Zimu Tian
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Contributions

Y.Z. conceived and supervised the project; H.Y. and Y.Z. designed the research; H.Y. performed technology development, mechanistic study, analytical characterization and clinical validation; H.Y. and Z.T. conducted the LFA experiments; H.Y. and N.H. constructed the POC device; H.Y. and Y.W. performed isolation and NTA analysis of EVs; S.H. conducted cell culture and provided culture media; S.H. helped with TEM analysis and was involved in experiment design and discussion; S.J.H. assisted in the clinical studies; H.Y. and Y.Z. analysed the data and wrote the paper. All authors edited the paper.

Corresponding author

Correspondence to Yong Zeng.

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Competing interests

H.Y. and Y.Z. are co-inventors on a United States provisional patent application (63/313,870) based on this work. Y.Z. holds equity interest in Clara Biotech and serves on its scientific advisory board. The other authors declare no competing interests.

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Nature Biomedical Engineering thanks Ruijie Deng and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Yan, H., Wen, Y., Tian, Z. et al. A one-pot isothermal Cas12-based assay for the sensitive detection of microRNAs. Nat. Biomed. Eng 7, 1583–1601 (2023). https://doi.org/10.1038/s41551-023-01033-1

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