Elsevier

Analytica Chimica Acta

Volume 1265, 18 July 2023, 341278
Analytica Chimica Acta

Highly sensitive and facile microRNA detection based on target triggered exponential rolling-circle amplification coupling with CRISPR/Cas12a

https://doi.org/10.1016/j.aca.2023.341278Get rights and content

Highlights

  • ā€¢

    The T-ERCA/Cas12a assay can achieve the ultrasensitive and facile detection.

  • ā€¢

    This system has higher amplification efficiency than single amplification.

  • ā€¢

    An excellent limit of detection down to 0.31Ā fM.

  • ā€¢

    It shows good application ability for assessing miRNA levels in different cells.

Abstract

MicroRNAs (miRNAs) play a crucial role in the regulation of gene expression and have been implicated in many diseases. Herein, we develop a target triggered exponential rolling-circle amplification coupling with CRISPR/Cas12a (T-ERCA/Cas12a) system, which can achieve the ultrasensitive detection with simple operation and no annealing procedure. In this assay, T-ERCA combines the exponential amplification with rolling-circle amplification by introducing a dumb-bell probe with two enzyme recognition sites. miRNA-155 targets are activators that trigger exponential rolling circle amplification to produce large amounts of ssDNA, which is then recognized by CRISPR/Cas12a for further amplification. Compared with single EXPAR or RCA combined with CRISPR/Cas12a, this assay shows higher amplification efficiency. Therefore, benefiting from the excellent amplification effect of T-ERCA and the high recognition specificity of CRISPR/Cas12a, the proposed strategy shows a wide detection range from 1Ā fM to 5Ā nM with a LOD (limit of detection) down to 0.31Ā fM. Moreover, it shows good application ability for assessing miRNA levels in different cells, indicating that the T-ERCA/Cas12a may provide a new guidance for molecular diagnosis and clinical practical application.

Introduction

MicroRNAs (miRNAs) are small noncoding RNAs consisting of approximately 20ā€“25 nucleotides. As a kind of transcription regulation factors, miRNAs perform post-transcriptional repression by binding to the 3ā€²UTR of target in the seed region, which is considered as a typical mode of miRNA-mediated gene regulation [[1], [2], [3]]. And they play their biological functions mainly by participating in regulating cell differentiation, apoptosis, proliferation, signal transduction and other biological processes [4]. Recent researches prove that abnormal expression of miRNA is strongly linked to the initiation and occurrence of human cancers [5]. Therefore, miRNA has been recognized as an effective biomarker for the diagnosis and prognosis of cancer. Developing rapid and accurate miRNAs detection platforms is not only helpful for cancer treatment, but also for the prevention of malignant diseases.

For miRNA detection, the main challenges are the short length of miRNA, low sequence homology and the abundance of family members [6,7]. However, the commonly used nucleic acid detection techniques such as northern blot [8], qRT-PCR [9], and DNA microarrays [10] require sophisticated experimental procedures and expensive instruments with long detection time and low sensitivity [11,12]. These problems may limit their point-of-care diagnostic applications. Recently, clustered regularly interspaced short palindromic repeats and its associated protein (CRISPR/Cas) system has aroused extensive concern in the field of disease diagnosis, especially when several Cas effectors with trans cleavage activity are discovered, such as Cas12, Cas13 and Cas14 [[13], [14], [15]]. The collateral cleavage activity of these proteins can be activated under the guidance of specific crRNA recognition and then indiscriminately cleats nearby single-stranded RNA (ssRNA) or single-stranded DNA (ssDNA) thousands of times per second [16,17]. These characteristics endow the detection of CRISPR/Cas system with high recognition specificity, self-amplification effect, simple operation, and rapid speed [18,19]. Among CRISPR/Cas systems, the CRISPR/Cas12a can recognize both dsDNA (double-stranded DNA) and ssDNA to perform its trans-cleavage activity. And ssDNA recognition does not require a PAM site [20,21]. Based on these properties, a variety of CRISPR/Cas12a-based detection strategies combined with isothermal nucleic acid amplification have emerged, such as opvCRISPR (RT-LAMP-CRISPR/Cas12a) [22], Cas12a-SCR (RCA-Cas12a) [23], RPA-Cas12A-FS [24], etc. The CRISPR/Cas system can effectively improve the detection sensitivity by combining with isothermal amplification technology. Meanwhile, compared with the traditional polymerase chain reaction (PCR), isothermal amplification has the characteristics of mild reaction conditions and high amplification efficiency [25,26], and has better adaptability with CRISPR/Cas, which is suitable for development as a clinical POCT detection instrument. However, most of the CRISPR/Cas-mediated detections only combine with a single amplification method, which could not achieve ideal results [27]. Therefore, integrating the characteristics of different isothermal amplification techniques to develop novel CRISPR-based detection platforms may be an ideal method.

In this work, a novel fluorescent approach based on target triggered exponential rolling-circle amplification coupling with CRISPR/Cas12a (T-ERCA/Cas12a) is developed for sensitive detection of miRNA. Herein, a dumb-bell probe with two enzyme recognition sites and two target recognition domains is designed as amplification template to trigger the T-ERCA reaction, which can generate a large amount of ssDNA. The obtained ssDNA can activate the trans-cleavage activity of Cas12a for fluorescence signal output. Based on the above principles, the proposed strategy shows high sensitivity, selectivity, repeatability, and stability. As low as 0.31Ā fM of miRNA-155 can be detected. Compared with single EXPAR or RCA combined with CRISPR/Cas12a, this assay shows higher amplification efficiency. Moreover, the T-ERCA/Cas12a system can be used to identify the miRNA level in different cells, and the results are consistent with those of quantitative reverse transcription polymerase chain reaction (qRT-PCR), which proves the reliability of the proposed method.

Section snippets

Material and reagents

HiScribe T7 High Efficiency RNA Synthesis Kit, Exonuclease I (Exo I), Exonuclease III (Exo III), T4 DNA ligase, Phi29 polymerase, and Nt.BbvCI were obtained from New England BioLabs (Beijing, China). The RNase-free H2O, the miRNA purification kit and RNAsimple Total RNA Kit (DP419) were available from TIANGEN (Beijing, China). Cas12a (Cpf1) was obtained from Guangzhou Meige Biological Technology. miRNAFirst Strand cDNA Synthesis (Stem-loop Method), 2X SG Fast qPCR Master Mix, TE buffers,

Principle of the T-ERCA/Cas12a system

The mechanism of the target triggered exponential rolling-circle amplification coupling with CRISPR/Cas12a (T-ERCA/Cas12a) system is shown in Scheme 1. To achieve a simple and efficient detection strategy, we first prepare the dumb-bell probe (DP), which is the amplification template for next T-ERCA/Cas12a. The DP is designed with two restriction sites and synthesized by T4 DNA ligase, and then the product is treated with EXO I and EXO III to digest the unlinked probe (Scheme 1A). The prepared

Conclusion

In summary, inspired by the characteristics of the traditional exponential amplification and rolling-circle amplification technology, we develop a novel isothermal amplification strategy, T-ERCA/Cas12a system, for target miRNA-155 detection. By introducing a dumb-bell probe with two enzyme recognition sites as templates, the sensitivity and accuracy of detection were effectively improved. And the amplified ssDNA of T-ERCA reaction enables the collateral cleavage activity of Cas12a to cleft the

CRediT authorship contribution statement

Shiying Zhou: Writing ā€“ original draft, The major experiments and manuscript writing were finished by, All authors who have made substantial contributions to the work are listed in the manuscript. Human Sun: Some Cell culture and measurements were finished by, All authors who have made substantial contributions to the work are listed in the manuscript. Jiangbo Dong: Some Cell culture and measurements were finished by, All authors who have made substantial contributions to the work are listed in

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (NO. 81772290), Chongqing science and technology commission (cstc2021jcyj-msxmX0608), Graduate Research and Innovation Foundation of Chongqing, China (CYB22072), Fundamental Research Funds for the Central Universities (2022CDJYGRH-013), Chongqing Natural Science Foundation (CSTB2022NSCQ-BHX0727), Sichuan Science and Technology Program (2022YFSY0013), Chongqing Graduate Tutor Team Construction Project, and the sharing

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