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

MicroRNAs (miRNAs) are small noncoding RNAs consisting of approximately 20–25 nucleotides. As a kind of transcription regulation factors, miRNAs perform post-transcriptional repression by binding to the 3′UTR 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 0.31 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.

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