Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2025 Jun;41(6):867-876.
doi: 10.1007/s44211-025-00755-3. Epub 2025 Apr 21.

Ultra-sensitive biosensor detection of microRNA based on the CRISPR/Cas12a system and exonuclease-assisted target recycling signal amplification

Xing Yang  1 , Yu Luo  1 , Chunhua Su  2 , Zhimei Huang  1 , Yafang Tang  3 , Liwen Zhang  4
Affiliations

Ultra-sensitive biosensor detection of microRNA based on the CRISPR/Cas12a system and exonuclease-assisted target recycling signal amplification

Xing Yang et al. Anal Sci. 2025 Jun.

Abstract

MicroRNAs (miRNAs) are essential regulators of gene expression and are significantly involved in both preventing and treating a range of diseases. To that end, we developed an ultra-sensitive detection method for miRNA-141 by integrating exonuclease-assisted target recycling signal amplification with the CRISPR/Cas12a system. This method employs a variable hairpin probe (HP) designed to hybridize with miRNA, which, under the action of exonuclease III (ExoIII), cleaves the hairpin probe and triggers target recycling signal amplification. This results in the formation of output DNAs (ODs) containing multiple repeat sequences. The CRISPR/Cas12a system identifies these repeated sequences in ODs through its crRNA component, which in turn triggers the trans-cleavage function of the Cas12a/crRNA complex. It leads to the cleavage of a fluorescently quenched reporter probe. Consequently, this process restores fluorescence, producing a significantly enhanced fluorescent signal that facilitates the detection of miRNA-141, achieving a detection threshold down to 62 fM. This detection approach can specifically differentiate miRNA-141 from other confounding substances and has effectively identified low concentrations of miRNA-141 in actual sample human serum and diverse cancer cell lysates, showcasing its capability for tracing various nucleic acid biomarkers at minimal levels.

Keywords: CRISPR/Cas12a; ExoIII; MiRNA-141; Target recycling signal amplification.

PubMed Disclaimer

Conflict of interest statement

Declarations. Conflict of interest: The authors declare no competing interests.

Similar articles

See all similar articles

References

    1. E.S. Lander, The heroes of CRISPR. Cell 164(1–2), 18–28 (2016). https://doi.org/10.1016/j.cell.2015.12.041 - DOI - PubMed
    1. M. Wang, R. Zhang, J. Li, CRISPR/cas systems redefine nucleic acid detection: principles and methods. Biosens. Bioelectron. 165, 112430 (2020). https://doi.org/10.1016/j.bios.2020.112430 - DOI - PubMed - PMC
    1. J. Liu, S. Srinivasan, C.Y. Li, I.L. Ho, J. Rose, M. Shaheen et al., Pooled library screening with multiplexed Cpf1 library. Nat. Commun. 10(1), 3144 (2019). https://doi.org/10.1038/s41467-019-10963-x - DOI - PubMed - PMC
    1. F. Wang, Y. Hao, Q. Li, J. Li, H. Zhang, X. Zhang et al., Programming PAM antennae for efficient CRISPR-Cas9 DNA editing. Sci. Adv. 6(19), eaay9948 (2020). https://doi.org/10.1126/sciadv.aay9948 - DOI - PubMed - PMC
    1. R. Zhu, H. Jiang, C. Li, Y. Li, M. Peng, J. Wang et al., CRISPR/Cas9-based point-of-care lateral flow biosensor with improved performance for rapid and robust detection of Mycoplasma pneumonia. Anal. Chim. Acta 1257, 341175 (2023). https://doi.org/10.1016/j.aca.2023.341175 - DOI - PubMed

LinkOut - more resources