CRISPR-Cas12a enhanced rolling circle amplification method for ultrasensitive miRNA detection
Introduction
MicroRNAs (miRNAs) are small, non-coding RNAs that paly vital roles in regulating gene expression and related pathological process [1], [2]. Abnormal expressions of miRNAs are closely related to many diseases including retinal disorder, neurodegenerative diseases, cardiovascular disease and cancer [3], [4]. The in vitro detection and in situ imaging of miRNAs display great significance for early diagnosis of many diseases, especially cancers [5], [6]. Exosomes, as nanosized vesicles released from body cells, are reported to contain several different biomolecules including proteins, lipids, and both miRNAs and noncoding RNAs [7], [8]. Exosomes are also considered as the important cell-cell communication medium since they can release the contents, particularly miRNAs, to both neighboring and distal cells [9]. Thus, exosomal miRNA detection methods are of great importance to not only the diagnosis of various diseases, but also the pathologic studies. Even though, miRNA detection methods need continuously improvement to be with higher sensitivity and accuracy due to its unique characteristics of miRNAs, such as the short size (about 22 nucleotides), low abundance, high sequence homology between family members, and vulnerable degradation [10], [11], [12].
Many traditional miRNA detection methods, such as real-time quantitative polymerase chain reaction (qRT-PCR), northern blotting and microarray are reported to achieve sensitive miRNAs detection in solutions or in cell lysates [13], [14], [15]. Even though, they are also criticized for time-consuming steps, complex and expensive, impeding their wide applications. In recent, a variety of new strategies for in vitro miRNA detection with good sensitivity have been developed based on various signal amplification mechanisms, such as rolling circle amplification (RCA) [16], [17], hybridization chain reaction (HCR), recombinase polymerase amplification (RPA) [18], [19], [20], [21]. These free-PCR methods are designed elegantly and have made some progress to some extent. In particular, RCA assay has been attracted more attention due to its stable signal output and easy-to-operate characteristic. RCA has emerged as a highly specific isothermal gene amplification approach that performed at a constant temperature (at 30 °C or even room temperature) with thermally stable DNA polymerase and without sophisticated instruments [21], [22], [23]. In addition, RCA can convert short-length RNA in to single-strand DNA (ssDNA), which is much more stable in a complicated environment such as serum. Nevertheless, the sensitivity of RCA-based nucleic acid detection strategies needs to be improved as it is widely reported to be with merely one hundred times target amplification, which fails to beat the recently introduced miRNA detection methods and to meet clinical application requirements [24], [25].
CRISPR/Cas (Clustered regularly interspaced short palindromic repeats), as an RNA-based acquired immune system of most bacteria and archaea, has become a powerful gene editing tool and is widely used in gene function research, gene modification and treatment [26], [27]. Among all the Cas enzymes discovered, CRISPR-Cas12a (Cpf1), which is capable of binding and cutting both dsDNA (double chain DNA) and ssDNA target under the guidance of sgRNA and triggering the following trans-cleavage activity, attracts a multitude of attention for nucleic acid detection [28], [29], [30]. Recently, a new CRISPR-derived miRNA detection strategy has been proposed based on Cas12a self-powered and rolling circle transcription-unleashed real-time crRNA recruiting [31], [32]. Even though a favorable performance is obtained, the single-strand RNA (ssRNA) product from rolling circle transcription process is more fragile to complicated conditions compared with ssDNA products.
Herein, we propose a novel CRISPR-Cas12a enhanced RCA method for miRNA detection by integrating the RNA to ssDNA conversion ability of RCA and trans-cleavage of CRISPR-Cas12a. In the proposed method, target miRNA can be amplified into long ssDNA chain, which is composed by padlock complementary repeat sequences. The obtained padlock complementary repeat sequences can thereby be recognized by CRISPR-Cas12a and thus trigger its trans-cleavage towards surrounding DNA reporters, whose two terminals are labeled with a fluorophore (Cy3) and a corresponding quenching group (BHQ1) to induce fluorescence resonance energy transfer (FRET). After optimizing the operating parameters, we have investigated the performance of the proposed method with commonly used quantitative polymerase chain reaction (RT-qPCR) method in the detection of exosomal miRNA-21 from both cell culture supernatant and clinical samples with extraordinary sensitivity and obtained a favorable consistency. From our perspective, the proposed CRISPR-Cas12a enhanced RCA method exhibit a remarkable improvement towards conventional RCA base method and show a promising prospect for disease diagnosis.
Section snippets
Principle of the proposed method
The working mechanism of the proposed exosomal miRNA sensing strategy was depicted in Scheme 1. To realize the sensitive in vitro exosomal miRNA detection, we divided the whole biosensing process into two steps: RCA initiated signal amplification and CRISPR-Cas12a based specific cleavage. For the amplification step, the target miRNA could simultaneously bind to the two terminals of padlock, making the 3′- and 5′-ends of padlock adjacent and producing cyclized padlock catalyzed by T4 DNA ligase.
Conclusions
We reported here a novel miRNA detection method by integrating the advantages of powerful RCA and CRISPR-Cas12a-mediated specific trans-cleavage. With the Cas12a enhanced signal amplification, the proposed strategy exhibited an improved sensitivity towards the in vitro exosomal miRNA detection. Different from most of the conventional RCA-based sensing system, the method was easy-to-operated due to the wash free character. Thus, we believe that the method not only show a promising prospect for
Reagents and apparatus
All oligonucleotide sequences in Table S1 were purchased and purifies by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd (Shanghai, China). EnGen ® Lba Cas12a enzymes were purchased from BioLabs Tech Co., Ltd (Beijing, China). T4 DNA ligase, T4 polynucleotide kinase, Phi29 DNA polymerase and NTP Mix were purchased from New England BioLabs (MA, U.S.A.). Acryl/Bis 30% Solution, Ammonium Per Sulfate, N,N,N′,N′-Tetramethylethylenediamine were purchased from Shanghai Sangon
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.
Acknowledgements
This work was financially supported by the National Natural Science Foundation of China (Grant Nos. 81873279, 81473628), Shanghai Science and Technology Commission Hospital Guidance Project (No. 19401935200); 4) Shanghai Municipal Health Planning Commission Clinical Special Project (No. 201840325).
Ethical approval
Approval from Southwest Hospital.
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These authors contributed equally to this work.