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ORIGINAL RESEARCH article

Front. Bioeng. Biotechnol.
Sec. Biosensors and Biomolecular Electronics
Volume 12 - 2024 | doi: 10.3389/fbioe.2024.1444908

An ultra-sensitive suboptimal protospacer adjacent motif enhanced rolling circle amplification assay based on CRISPR/Cas12a for detection of miR-183

Provisionally accepted
Zhiquan Lu Zhiquan Lu 1Shijing Wang Shijing Wang 2Ping Li Ping Li 3Huasheng Yang Huasheng Yang 4Sanyang Han Sanyang Han 3Shaochong Zhang Shaochong Zhang 2*Lan Ma Lan Ma 1,3,5*
  • 1 Tsinghua-Berkeley Shenzhen Institute, Tsinghua University, Shenzhen, China
  • 2 Shenzhen Eye Hospital, Shenzhen, China
  • 3 Institute of Biopharmaceutical and Health Engineering, Shenzhen International Graduate School, Tsinghua University, Shenzhen, China
  • 4 State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, Guangdong Province, China
  • 5 Shenzhen Bay Laboratory, Shenzhen, Guangdong, China

The final, formatted version of the article will be published soon.

    MicroRNAs (miRNAs) have been recognized as promising diagnostic biomarkers for Diabetic Retinopathy (DR) due to their notable up-regulation in individuals with the condition. However, the development of highly sensitive miRNAs assays for the rapid diagnosis of DR in clinical settings remains a challenging task. In this study, we introduce an enhanced CRISPR/Cas12a assay, leveraging suboptimal PAM (sPAM)-mediated Cas12a trans-cleavage in conjunction with rolling circle amplification (RCA). We found that sPAM performed better than canonical PAM (cPAM) in the detection of Cas12a-mediated ssDNA detection at low concentrations, and by replacing cPAM with sPAM in the padlock template of RCA, ultra-high sensitivity can be achieved, with a detection limit of 0.40 aM within 25 minutes and a linear range spanning from 1 aM to 1 pM. Notably, our assay exhibits exceptional specificity in detecting miR-183 from other miRNAs. Furthermore, we validate the applicability of our assay for the sensitive detection of miR-183 in clinical serum samples. This study introduces a groundbreaking assay with excellent performance through a simple modification, which not only addresses existing diagnostic challenges, but also opens exciting new avenues for clinical diagnosis in the realm of Diabetic Retinopathy.

    Keywords: Diabetic retinopathy1, CRISPR/Cas12a2, microRNA3, rolling circle amplification4, fluorescence5

    Received: 06 Jun 2024; Accepted: 05 Sep 2024.

    Copyright: © 2024 Lu, Wang, Li, Yang, Han, Zhang and Ma. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

    * Correspondence:
    Shaochong Zhang, Shenzhen Eye Hospital, Shenzhen, 518040, China
    Lan Ma, Tsinghua-Berkeley Shenzhen Institute, Tsinghua University, Shenzhen, China

    Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.