Highly sensitive and facile microRNA detection based on target triggered exponential rolling-circle amplification coupling with CRISPR/Cas12a
Graphical abstract
Introduction
MicroRNAs (miRNAs) are small noncoding RNAs consisting of approximately 20ā25 nucleotides. As a kind of transcription regulation factors, miRNAs perform post-transcriptional repression by binding to the 3ā²UTR 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
References (38)
- et al.
Classical and noncanonical functions of miRNAs in cancers
Trends Genet.
(2022) - et al.
Targeting miRNAs by natural products: a new way for cancer therapy
Biomed. Pharmacother.
(2020) - et al.
PCDetection: PolyA-CRISPR/Cas12a-based miRNA detection without PAM restriction
Biosens. Bioelectron.
(2022) - et al.
NEase-based amplification for detection of miRNA, multiple miRNAs and circRNA
Anal. Chim. Acta
(2021) - et al.
Palindromic hyperbranched rolling circle amplification enabling ultrasensitive microRNA detection
Chem. Commun.
(2022) - et al.
Research advances in the detection of miRNA
Journal of Pharmaceutical Analysis
(2019) - et al.
microRNA-guided diagnostics in clinical samples
Best Pract. Res. Clin. Endocrinol. Metabol.
(2016) - et al.
Detection methods for microRNAs in clinic practice
Clin. Biochem.
(2013) - et al.
CRISPR/cas systems redefine nucleic acid detection: principles and methods
Biosens. Bioelectron.
(2020) - et al.
CRISPR-Cas12a-assisted nucleic acid detection
Cell Discovery
(2018)
opvCRISPR: one-pot visual RT-LAMP-CRISPR platform for SARS-cov-2 detection
Biosens. Bioelectron.
RPA-Cas12a-FS: a frontline nucleic acid rapid detection system for food safety based on CRISPR-Cas12a combined with recombinase polymerase amplification
Food Chem.
Point-of-care CRISPR/Cas nucleic acid detection: recent advances, challenges and opportunities
Biosens. Bioelectron.
Novel and simple electrochemical biosensor monitoring attomolar levels of miRNA-155 in breast cancer
Biosens. Bioelectron.
CRISPR-Cas12a enhanced rolling circle amplification method for ultrasensitive miRNA detection
Microchem. J.
Ratiometric electrochemical detection of miRNA based on DNA nanomachines and strand displacement reaction
Microchim. Acta
Construction of an ultrasensitive electrochemical sensing platform for microRNA-21 based on interface impedance spectroscopy
J. Colloid Interface Sci.
Visual detection of heart failure associated MiRNA with DSN enzyme-based recycling amplification strategy
RSC Adv.
Northern blotting analysis of microRNAs, their precursors and RNA interference triggers
BMC Mol. Biol.
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