A CRISPR-Cas13a trans-cleavage-based method and kit for quantitative real-time detection of miRNAs.

Abstract

This invention provides a quantitative fluorescence detection method and kit for miRNA based on CRISPR-Cas13a trans-cleavage. The detection method involves forming a double-stranded complex between the target miRNA and a specific crRNA, activating the trans-nuclease activity of the Cas13a protein, non-specifically cleaving a fluorescently labeled RNA reporter molecule, and releasing a fluorescent signal. Quantitative detection of miRNA is achieved by real-time monitoring of fluorescence intensity and combining it with a standard curve. This detection method eliminates the need for reverse transcription and PCR amplification, completing the detection within 20 to 40 minutes at a constant temperature of 37°C; the detection sensitivity reaches 0.1 fM, with a linear range of 0.1 fM–100 pM; it can distinguish single-base differences; and it is suitable for detecting various trace samples such as plasma, tissue, and cells. This invention provides a new technical solution for rapid and accurate miRNA detection, with broad application prospects in early tumor diagnosis and treatment monitoring.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid molecular detection technology, particularly to the fields of molecular diagnostics and clinical laboratory medicine, and specifically to a method for quantitative detection of microRNA based on the trans-cleavage activity of CRISPR-Cas13a nuclease and a corresponding detection kit. Background Technology

[0002] MicroRNAs are a class of endogenous non-coding single-stranded RNA molecules, approximately 18–25 nucleotides in length, that regulate gene expression at the posttranscriptional level by binding to the 3' untranslated region of target gene mRNA. Numerous studies have shown that circulating miRNAs are ideal disease diagnostic biomarkers and drug targets; therefore, accurate quantitative detection of miRNAs has significant clinical value for early disease diagnosis, subtyping, efficacy monitoring, and prognostic assessment.

[0003] Currently, miRNA detection methods mainly include Northern blot hybridization, microarray chips, and real-time quantitative PCR. Northern blot hybridization has low sensitivity and requires a large number of samples, so it is rarely used now. Microarray chips are suitable for high-throughput expression profiling analysis, but their specificity and accuracy are limited, and the results need further verification. Real-time quantitative PCR is currently the most commonly used method for miRNA quantitative detection, but it has the following limitations: (1) It generally requires a reverse transcription step. Since miRNAs are short and lack poly(A) tails, special reverse transcription strategies are required, such as stem-loop primers and tailing methods. Chinese patent CN106148500B discloses a method that combines poly(A) tailing reaction and reverse transcription reaction into one, but it still requires two enzymatic reactions, which is relatively complicated to operate, and the reverse transcription efficiency is affected by various factors, which may introduce systematic errors. (2) The PCR amplification step takes a long time, usually requiring 2-4 hours to complete the detection, which is difficult to meet the needs of rapid diagnosis. (3) miRNA sequences have high similarity, and family members often differ by only 1-2 bases. Traditional PCR methods have limited ability to distinguish single-base differences, which may lead to cross-reactions and affect detection specificity. (4) PCR methods have high requirements for the operating environment and are prone to aerosol contamination, resulting in false positive results. (5) For extremely small samples such as single cells and exosomes, the multi-step operation of reverse transcription and PCR may lead to sample loss and detection errors.

[0004] In recent years, the CRISPR-Cas system has received widespread attention as a next-generation gene editing tool. Unlike the Cas9 and Cas12 systems, which primarily cleave DNA, Cas13 is a type of CRISPR effector protein that specifically targets RNA. After recognizing target RNA under the guidance of crRNA, the Cas13 protein not only cleaves the target RNA but also activates its trans-cleavage activity, i.e., non-specifically cleaving other surrounding RNA molecules. This "bystander effect" can be used for nucleic acid detection. Existing CRISPR-based nucleic acid detection technologies mainly focus on DNA detection, such as the SHERLOCK and DETECTR platforms. However, these technologies still require reverse transcription to convert RNA into DNA before detecting RNA, failing to fully utilize the advantage of Cas13's direct RNA recognition. Currently, research on CRISPR-Cas13 detection methods for short-chain RNAs such as miRNAs is relatively limited, lacking system optimization and clinical validation. Therefore, there is an urgent need in this field to develop a new, rapid, highly sensitive, and highly specific method for the quantitative detection of miRNAs that requires no reverse transcription or PCR amplification to meet the needs of clinical diagnosis and scientific research. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a quantitative fluorescence detection method and kit for miRNA based on CRISPR-Cas13a trans-cleavage. This detection method does not require reverse transcription and PCR amplification steps and can be completed rapidly under isothermal conditions. It has the advantages of high sensitivity, strong specificity, simple operation and rapid detection. It is suitable for a variety of sample types and has broad application prospects in clinical disease diagnosis and biomedical research.

[0006] To achieve the above objectives, the present invention provides the following technical solution: The first approach of this invention provides a method for quantitative fluorescence detection of miRNA based on CRISPR-Cas13a trans-cleavage, comprising the following steps: The first step is to provide a sample containing the miRNA to be tested; The second step involves mixing the sample to be tested with the detection reagents to construct a reaction system. This reaction system includes Cas13a protein at a concentration of 20–200 nM, specific crRNA at a concentration of 20–200 nM, fluorescently labeled RNA reporter molecules at a concentration of 5–50 nM, and a reaction buffer. The specific crRNA contains a guide sequence complementary to the target miRNA, with a guide sequence length of 18–28 nucleotides, and is either completely complementary to the target miRNA or allows for mismatches of no more than 2 bases. The reaction buffer consists of 20 mM HEPES pH 7.5, 10 mM MgCl2, and 100 mM KCl. The third step is isothermal incubation and real-time monitoring of fluorescence signals: The reaction system is incubated at a constant temperature of 30-42℃ for 15-60 minutes. During the incubation, the miRNA to be tested forms a double strand with the crRNA, which activates the trans-cleavage activity of Cas13a, cleaving the fluorescently labeled RNA reporter molecule and releasing a fluorescence signal. The instrument collects fluorescence signals every 30-120 seconds and records the time point or cycle number when the fluorescence signal reaches the set threshold. The fourth step is data analysis and quantitative calculation: a standard curve is established in advance using miRNA standards of a series of concentrations. The fluorescence signal collected during the isothermal incubation process is compared with the standard curve to calculate the concentration of the miRNA to be tested in the sample.

[0007] Furthermore, the above detection method allows for the addition of multiple crRNAs targeting different miRNAs and RNA reporter molecules labeled with different fluorescent groups to the same reaction system, enabling the simultaneous detection of 2 to 6 miRNAs with a peak emission wavelength interval of ≥30 nm for the fluorescent groups.

[0008] Preferably, in the above detection method, the reaction system includes Cas13a protein at a concentration of 50 nM, specific crRNA at a concentration of 50 nM, and fluorescently labeled RNA reporter molecule at a concentration of 10 nM; the reaction system is incubated at a constant temperature of 37°C for 30 minutes.

[0009] In the first step of the above detection method, the sample can be a purified RNA solution after RNA extraction, or a sample that has undergone simple lysis treatment, including but not limited to blood, plasma, serum, urine, saliva, cerebrospinal fluid, tissue homogenate, cell lysate, exosome solution, or single-cell lysate.

[0010] In the second step of the above detection method, the specific crRNA contains a guide sequence complementary to the target miRNA, with a length of 18-28 nucleotides, preferably 20-24 nucleotides, and most preferably 22 nucleotides. The crRNA sequence is designed to be completely complementary to the target miRNA or to allow mismatches of no more than 2 bases to ensure specific recognition.

[0011] In the second step of the above detection method, the fluorescently labeled RNA reporter molecule is designed based on the fluorescence resonance energy transfer principle, with a structure of 5'-fluorescent group-ribonucleotide sequence-quencher group-3'. In its intact state, the quencher group and the fluorescent group are very close, and the fluorescence is quenched. When the reporter molecule is cleaved by Cas13a, the fluorescent group and the quencher group separate, releasing a fluorescent signal. Common fluorescent dyes such as FAM, HEX, ROX, and Cy5 can be used for the fluorescent group, and BHQ1, BHQ2, and DABCYL can be used for the quencher group. The ribonucleotide sequence of the reporter molecule preferably consists of 4-10 uracil nucleotides because Cas13a has a high cleavage efficiency for U-rich sequences, and the optimal ribonucleotide sequence of the reporter molecule consists of 5 uracil nucleotides.

[0012] Preferably, the concentrations of each component in the reaction system are finely optimized: the Cas13a protein concentration is preferably 50 nM; the specific crRNA concentration is preferably 50 nM; the fluorescently labeled RNA reporter molecule concentration is preferably 10 nM; the total reaction volume of the reaction system can be set according to actual needs, usually 10-50 μL, preferably 20 μL.

[0013] The reaction buffer consisted of: 20 mM HEPES buffer at pH 7.5; 10 mM magnesium chloride; and 100 mM potassium chloride. This buffer system was obtained through experimental optimization and can simultaneously ensure the stability of the Cas13a protein, effective hybridization of crRNA and miRNA, and optimal cleavage activity.

[0014] The third step of the above detection method is as follows: (1) Place the mixed reaction system in a real-time fluorescence PCR instrument, microplate reader, or dedicated real-time fluorescence detector, and incubate it under constant temperature conditions. The reaction temperature is 30-42℃, preferably 37℃; the reaction time is 15-60 minutes, preferably 20-40 minutes. During the incubation process, the miRNA to be tested forms a double-stranded complex with crRNA, which activates the trans-nuclease activity of Cas13a protein, enabling it to non-specifically and rapidly cleave the fluorescently labeled RNA reporter molecule in the reaction system and release a fluorescence signal; (2) During the reaction, the instrument collects fluorescence signals at certain time intervals, usually once every 30 seconds to 2 minutes. The fluorescence intensity gradually increases as the reporter molecule is cleaved, showing a typical S-shaped growth curve. Record the time point or cycle number when the fluorescence signal reaches the set threshold, similar to the Ct value concept in quantitative PCR, and define it as the threshold time Tt.

[0015] The fourth step of the above detection method involves establishing a standard curve using miRNA standards at a series of concentrations. This curve is plotted with the logarithm of the miRNA concentration on the x-axis and the threshold time (Tt) on the y-axis, and a linear fit is performed. The miRNA concentration of the sample to be tested can be calculated from the standard curve based on its Tt value.

[0016] The detection principle of the method of this invention is based on the following scientific mechanism: The CRISPR-Cas13a system consists of two parts: the Cas13a effector protein and crRNA. The crRNA contains a guide sequence complementary to the target RNA. When the crRNA forms a double strand with the target RNA, it induces a conformational change in the Cas13a protein, activating the RNase activity of its two HEPN domains. Activated Cas13a not only cleaves the recognized target RNA, but more importantly, it acquires trans-cleavage activity, meaning it can non-specifically and rapidly cleave surrounding single-stranded RNA molecules without sequence limitations.

[0017] In the detection system of this invention, the target miRNA binds to a specifically designed crRNA, activating the trans-cleavage activity of Cas13a. A pre-added fluorescently labeled RNA reporter molecule acts as a "bystander" RNA, undergoing rapid and extensive cleavage, leading to an exponential increase in the fluorescence signal. This signal amplification mechanism gives this method extremely high sensitivity; theoretically, a single miRNA molecule can trigger thousands of reporter cleavage events.

[0018] The method of this invention has the following outstanding substantive features and significant progress compared with the prior art: First, no reverse transcription step is required. Cas13a directly recognizes and cleaves RNA, avoiding the efficiency loss and bias in the reverse transcription process, simplifying the operation process, reducing sources of error, and making it particularly suitable for the detection of trace samples.

[0019] Second, no PCR amplification is required. The reaction is carried out under isothermal conditions, eliminating the need for a thermal cycler, thus reducing equipment requirements and avoiding aerosol contamination and non-specific amplification issues that may occur during PCR.

[0020] Third, the testing speed is fast. The entire testing process can be completed within 20-40 minutes, which is significantly shorter than the 2-4 hours of the traditional qRT-PCR method, meeting the needs of rapid diagnosis.

[0021] Fourth, extremely high sensitivity. Based on the trans-cleavage amplification effect of Cas13a, the detection sensitivity of the method of this invention can reach the level of 0.1 fM, which is equivalent to the detection of only about 60 miRNA molecules in each 20 μL reaction system. The sensitivity is equal to or better than that of the traditional qRT-PCR method.

[0022] Fifth, it has high specificity. The pairing of crRNA and miRNA follows a strict base complementarity principle, which can distinguish single-base differences. Experimental verification shows that for miRNA family members with only one base difference, the signal difference ΔTt of this method can reach more than 3 cycles, effectively avoiding cross-reaction.

[0023] Sixth, it has a wide linear range. The linear detection range of this method can span 6-7 orders of magnitude, from 0.1 fM to 100 pM or even higher, which can meet the detection needs of miRNAs with different abundances.

[0024] Seventh, it has broad sample applicability. This method can be used directly for bodily fluid samples such as plasma and serum, requiring only 5 μL of sample for detection; for single-cell samples, they can be directly added to the reaction system after simple lysis, without the need for complex RNA extraction and purification steps.

[0025] Eighth, it is easy to operate. The reaction system is simple to prepare, requiring only mixing of the components and incubation at a constant temperature. No multi-step operation is needed, making it easy to automate and apply at high throughput.

[0026] Ninth, it is cost-effective. Cas13a protein and crRNA can be produced on a large scale through recombinant expression and chemical synthesis without the need for expensive reverse transcriptase and DNA polymerase, making the cost controllable.

[0027] In a second approach, the present invention also provides a CRISPR-Cas13a trans-cleavage-based miRNA fluorescence quantitative detection kit, comprising the following core components: (1) Cas13a protein; (2) One or more specific crRNAs, said specific crRNAs containing a guide sequence complementary to the miRNA to be tested; (3) One or more fluorescently labeled RNA reporter molecules, wherein the structure of the fluorescently labeled RNA reporter molecule is 5'-fluorescent group-ribonucleotide sequence-quencher group-3', wherein the ribonucleotide sequence is composed of 4 to 10 uracils; multiple fluorescently labeled RNA reporter molecules designed for the simultaneous detection of multiple target miRNAs are RNA reporter molecules labeled with different fluorescent groups; (4) The reaction buffer consists of: 20 mM HEPES pH 7.5, 10 mM MgCl2, and 100 mM KCl; The miRNA to be tested is preferably selected from miR-21, miR-16, miR-155, miR-210, miR-122, let-7 family, and miR-200 family.

[0028] Furthermore, in the above kit, the Cas13a protein, specific crRNA, fluorescently labeled RNA reporter molecule, and reaction buffer are all individually packaged solutions; or the Cas13a protein, specific crRNA, fluorescently labeled RNA reporter molecule, and reaction buffer are mixed, and then trehalose and mannitol are added as protective agents, followed by lyophilization to produce a powder.

[0029] Furthermore, the above kit also includes: positive control miRNA standard, negative control solution, RNase inhibitor, nuclease-free water, miRNA standard of various concentrations, and optically clear reaction tubes or reaction plates.

[0030] The kit components are detailed below: First component: Cas13a protein.

[0031] Cas13a protein can be derived from various bacteria, including but not limited to *Leptotrichia shahii* and *Leptotrichia wadei*. The protein can be obtained through recombinant expression and purification using a prokaryotic expression system, with a purity ≥90%. If Cas13a protein is provided in solution form, the storage concentration is 0.5–5 μM, stored in a glycerol-containing buffer at -80°C to -20°C.

[0032] The second component: specific crRNA.

[0033] crRNA can be prepared through in vitro transcription or chemical synthesis. For common clinically relevant miRNAs such as miR-21, miR-16, and miR-155, the kit provides one or more pre-designed and synthesized specific crRNAs. For other miRNAs, customized crRNA design and synthesis services are available. If crRNA is provided in solution form, the storage concentration is 1-10 μM, and it should be stored at -80°C to -20°C.

[0034] The third component: fluorescently labeled RNA reporter molecules.

[0035] The reporter molecule was prepared by chemical synthesis and purified by HPLC with a purity ≥95%. The optimal sequence is 5'-fluorescent group-UUUUU-quencher group-3'. The kit provides one or more pre-designed fluorescently labeled RNA reporter molecules. If the fluorescently labeled RNA reporter molecules are provided in solution form, the storage concentration is 0.1-2 μM, and they should be stored at -20°C protected from light.

[0036] Fourth component: reaction buffer.

[0037] The reaction buffer consists of 20 mM HEPES pH 7.5, 10 mM MgCl2, and 100 mM KCl. It can be prepared as a 5× or 10× concentrate and stored at 4℃ or -20℃.

[0038] The kit may also include the following auxiliary components: Component 5: Positive control miRNA standard. This is a synthesized miRNA with the same sequence as the detection target, at a known concentration, used to verify the effectiveness of the reaction system.

[0039] Component 6: Negative control solution. A solution containing no miRNA, used to assess background signal.

[0040] Component 7: RNase inhibitor. Used to protect RNA in the sample and reaction system from degradation, at a concentration of 40 U / μL.

[0041] Component 8: Nuclease-free water. Used for diluting samples and preparing reaction systems.

[0042] Component 9: A series of miRNA standards at different concentrations for the standard curve. This includes 5–7 concentration gradients, ranging from 0.1 fM to 100 pM, used to establish a quantitative standard curve.

[0043] Component 10: Optically transparent reaction tubes or 96-well / 384-well reaction plates. Specifically designed for fluorescence detection; the material ensures accurate acquisition of fluorescence signals.

[0044] The kit also comes with a detailed instruction manual, including sample processing methods, reaction system preparation steps, instrument parameter settings, and data analysis procedures.

[0045] Depending on the application scenario, the reagent kit can be designed in different specifications: Basic type: Contains only the core components; users need to provide their own standards and controls.

[0046] Standard type: Contains core components, standards, controls and auxiliary reagents, and can be used directly.

[0047] High-throughput type: compatible with 96-well or 384-well plates, equipped with crRNAs of various clinically relevant miRNAs for expression profiling screening.

[0048] Ready-to-use: Cas13a protein, specific crRNA, fluorescently labeled RNA reporter molecule, and reaction buffer are premixed and lyophilized. Users only need to add the sample and water to start the reaction, simplifying the operation to the greatest extent.

[0049] A third aspect of this invention relates to the application of the aforementioned kit in disease diagnosis. Numerous studies have shown that abnormal expression of specific miRNAs is closely related to a variety of diseases and can serve as biomarkers for disease diagnosis, subtyping, efficacy monitoring, and prognostic assessment.

[0050] In the field of tumor diagnosis, the kit of this invention can be used to detect tumor-related miRNAs, such as miR-21, miR-155, let-7 family members, miR-200 family members, miR-134, miR-124, miR-122, and miR-375. Among these, miR-21 is highly expressed in various tumors, including liver cancer, lung cancer, gastric cancer, colorectal cancer, and pancreatic cancer; elevated levels in the blood can serve as an indicator for early tumor diagnosis and prognostic assessment. miR-155 is abnormally expressed in lymphoma, leukemia, and liver cancer, and is associated with tumor invasion and metastasis. Let-7 family members are downregulated in various tumors, including lung cancer, and have tumor-suppressive effects; their expression levels are correlated with patient prognosis. MiR-200 family members participate in the epithelial-mesenchymal transition process and are closely related to tumor metastasis. In the field of cardiovascular disease diagnosis, myocardial-specific miRNAs such as miR-208, miR-499, and miR-1 are released into the bloodstream during acute myocardial infarction and can serve as early diagnostic biomarkers, superior to traditional myocardial enzyme profile detection. In the field of neurological diseases, miR-134 and miR-124 are abnormally expressed in diseases such as Alzheimer's and Parkinson's, possessing diagnostic and disease progression monitoring value. In the field of metabolic diseases, miR-122 and miR-375 play regulatory roles in diseases such as diabetes and non-alcoholic fatty liver disease; changes in their blood levels can reflect the disease status.

[0051] This invention's kit is particularly suitable for liquid biopsy applications. Traditional tissue biopsies suffer from invasiveness and sampling limitations, while circulating miRNAs are stably present in bodily fluids such as blood and urine, allowing for sample acquisition through minimally invasive methods. This invention's method requires only 5 μL of plasma for detection and eliminates the need for complex RNA extraction and purification, making it suitable for routine clinical testing and dynamic monitoring.

[0052] This invention can also be extended to multiplex detection applications. By adding multiple crRNAs targeting different miRNAs and corresponding fluorescently labeled RNA reporter molecules to the same reaction system, the simultaneous detection of multiple miRNAs can be achieved. For example, using different fluorescence channels such as FAM, HEX, ROX, and Cy5, 2-6 miRNAs can be detected simultaneously in one reaction well, significantly improving detection efficiency and reducing sample and reagent consumption.

[0053] To further improve detection sensitivity, this invention also provides a signal amplification strategy. The target miRNA can be isothermally amplified before the Cas13a reaction, using methods such as rolling circle amplification or strand displacement amplification, increasing the miRNA copy number by 100-1000 times before Cas13a detection. This allows the detection limit to reach the atmolar level, achieving single-molecule detection capability.

[0054] The method of this invention can also be integrated with other technology platforms. For example, by combining it with microfluidic chip technology, automated sample processing and high-throughput detection can be achieved; by combining it with smartphones or portable fluorescence detection devices, point-of-care testing devices can be developed for on-site diagnosis and disease screening in resource-constrained areas. Attached Figure Description

[0055] Figure 1 This is a schematic diagram illustrating the miRNA detection principle based on CRISPR-Cas13a trans-cleavage of the present invention. The figure illustrates the complete process by which the target miRNA binds to the specific crRNA to form a double-stranded complex, activates the trans-cleavage activity of the Cas13a protein, and cleaves the fluorescently labeled RNA reporter molecule to release a fluorescent signal.

[0056] Figure 2 This is a standard curve diagram for detecting miR-21 using the method of the present invention; Using synthetic miR-21 standards at a series of concentrations ranging from 0.1 fM to 100 pM, a linear relationship between fluorescence threshold time and the logarithm of miRNA concentration was established. The linear fitting coefficient R² = 0.998, demonstrating that the method has good linearity and quantitative ability. Figure 3 The figure shows the experimental results of the specific detection of single-base differentially expressed miRNAs using the method of the present invention; Using a specific crRNA designed for let-7a as a detection probe, let-7a (the target) and its family members let-7b, let-7c (each differing from let-7a by one base), and let-7d (distinguishing from let-7a by multiple bases) were simultaneously detected at a detection concentration of 10 pM, along with the non-specific target miR-21 and a template-free blank control. The results showed that let-7a produced the earliest fluorescence response (TtTt=12.3 min), while let-7b (TtTt=15.8 min) and let-7c (TtTt=16.2 min) had threshold time lags of 3.5 min and 3.9 min, respectively, and let-7d (TtTt=18.5 min) had a lag of 6.2 min. miR-21 and the blank control showed no effective fluorescence signal throughout the detection process. The detection curves clearly separated at the threshold line, demonstrating that the method of this invention can effectively distinguish miRNA sequences with single-base differences and exhibits high sequence specificity. Figure 4 A comparison chart of miRNA detection results for different sample types; The figure shows the fluorescence curves of plasma samples, total RNA extracted from the same plasma sample, and synthetic miR-16 standard detected using the method of this invention. The Tt values ​​of the three are close, proving that the method can be directly used for plasma sample detection without the need for complicated RNA extraction steps. Figure 5 Figure showing the results of the single-cell level miRNA detection experiment; Using the method of this invention to detect miR-21 in a single HepG2 liver cancer cell, a clear fluorescence signal was successfully obtained, and the number of miR-21 copies in a single cell was calculated to be approximately 1200, proving that the method is suitable for single-cell analysis. Figure 6 This is a comparison and verification diagram between the method of the present invention and the traditional qRT-PCR method; The method of this invention and the gold standard qRT-PCR method were used to detect miR-21 in 20 plasma samples from clinical liver cancer patients. The detection results of the two methods were highly positively correlated, with a correlation coefficient r=0.96 and a relative deviation of <5%, which proves the accuracy and reliability of the method of this invention. Figure 7 Figure showing the results of the simultaneous detection of multiple miRNAs; In the same reaction system, three fluorescence channels, FAM, HEX and ROX, were used to simultaneously detect miR-21, miR-155 and miR-16. The fluorescence curves of the three channels did not interfere with each other, and accurate quantitative results were obtained for each channel. Detailed Implementation

[0057] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Experimental methods not specified in the embodiments are performed under conventional conditions or as recommended by the reagent supplier.

[0058] This invention provides a method and kit for quantitative fluorescence detection of miRNA based on CRISPR-Cas13a trans-cleavage. The principle of miRNA detection based on CRISPR-Cas13a trans-cleavage in this invention is as follows: Figure 1 As shown: The miRNA to be tested binds to the specific crRNA to form a double-stranded complex, which activates the trans-cleavage activity of the Cas13a protein, cleaving the fluorescently labeled RNA reporter molecule and releasing a fluorescent signal.

[0059] This invention relates to a quantitative fluorescence detection method for miRNA based on CRISPR-Cas13a trans-cleavage, characterized by the following steps: The first step is to provide a sample containing the miRNA to be tested; The second step involves mixing the sample to be tested with the detection reagents to construct a reaction system. This reaction system includes Cas13a protein at a concentration of 20–200 nM, specific crRNA at a concentration of 20–200 nM, fluorescently labeled RNA reporter molecules at a concentration of 5–50 nM, and a reaction buffer. The specific crRNA contains a guide sequence complementary to the target miRNA, with a guide sequence length of 18–28 nucleotides, and is either completely complementary to the target miRNA or allows for mismatches of no more than 2 bases. The reaction buffer consists of 20 mM HEPES pH 7.5, 10 mM MgCl2, and 100 mM KCl. The third step is isothermal incubation and real-time monitoring of fluorescence signals: The reaction system is incubated at a constant temperature of 30-42℃ for 15-60 minutes. During the incubation, the miRNA to be tested forms a double strand with the crRNA, which activates the trans-cleavage activity of Cas13a, cleaving the fluorescently labeled RNA reporter molecule and releasing a fluorescence signal. The instrument collects fluorescence signals every 30-120 seconds and records the time point or cycle number when the fluorescence signal reaches the set threshold. The fourth step is data analysis and quantitative calculation: a standard curve is established in advance using miRNA standards of a series of concentrations. The fluorescence signal collected during the isothermal incubation process is compared with the standard curve to calculate the concentration of the miRNA to be tested in the sample.

[0060] This invention also provides a miRNA fluorescence quantitative detection kit, which mainly includes the following four reagents: (1) Cas13a protein; (2) One or more specific crRNAs, said specific crRNAs containing a guide sequence complementary to the miRNA to be tested; (3) One or more fluorescently labeled RNA reporter molecules, wherein the structure of the fluorescently labeled RNA reporter molecule is 5'-fluorescent group-ribonucleotide sequence-quencher group-3', wherein the ribonucleotide sequence is composed of 4 to 10 uracils; multiple fluorescently labeled RNA reporter molecules designed for the simultaneous detection of multiple target miRNAs are RNA reporter molecules labeled with different fluorescent groups; (4) The reaction buffer consists of: 20 mM HEPES pH 7.5, 10 mM MgCl2, and 100 mM KCl.

[0061] The miRNA to be tested is preferably selected from miR-21, miR-16, miR-155, miR-210, miR-122, let-7 family, and miR-200 family.

[0062] The Cas13a protein, specific crRNA, fluorescently labeled RNA reporter molecule, and reaction buffer are all individually packaged solutions; or the Cas13a protein, specific crRNA, fluorescently labeled RNA reporter molecule, and reaction buffer are mixed, and then trehalose and mannitol are added as protective agents, followed by freeze-drying to produce a powder.

[0063] The detection kit also includes: positive control miRNA standard, negative control solution, RNase inhibitor, nuclease-free water, miRNA standard of various concentrations, and optically clear reaction tubes or reaction plates.

[0064] Example 1: Preparation and purification of Cas13a protein In this embodiment, recombinant Cas13a protein for miRNA detection was prepared.

[0065] The Cas13a gene from *Leptotrichia shahii* was cloned into the prokaryotic expression vector pET28a to construct a recombinant expression plasmid containing a His tag. The recombinant plasmid was transformed into *E. coli* BL21 competent cells, plated on LB agar plates containing kanamycin, and incubated overnight at 37°C.

[0066] A single colony was picked and inoculated into 5 mL of LB liquid medium containing kanamycin, and cultured overnight at 37°C with shaking to obtain the seed culture. The seed culture was then transferred 1:100 to 500 mL of LB medium and cultured at 37°C with shaking until OD reached. 600 When the concentration reaches 0.6-0.8, add IPTG to a final concentration of 0.5 mM to induce protein expression, and incubate overnight at 16°C with shaking.

[0067] Collect bacterial cells, resuspend in buffer A, sonicate to disrupt the cells, centrifuge at 12,000 rpm for 30 minutes, and collect the supernatant. Load the supernatant onto a pre-equilibrated nickel column, wash away non-specific binding proteins with buffer A containing 20 mM imidazole, and elute the target protein with buffer A containing 250 mM imidazole.

[0068] The elution peak was collected and identified by SDS-PAGE electrophoresis to confirm the expected size of the Cas13a protein band. The purified protein was dialyzed into storage buffer, and the protein concentration was determined to be 2.5 mg / mL by the BCA method. After aliquoting, the protein was stored at -80°C for later use.

[0069] The function of the Cas13a protein was verified by enzyme activity assays. A reaction system containing Cas13a protein, crRNA, target RNA, and a fluorescent reporter molecule was prepared and incubated at 37°C, with fluorescence signals monitored in real time. The results showed that the fluorescence intensity increased significantly over time, proving that the Cas13a protein is active.

[0070] Example 2: Optimization of Reaction Conditions This embodiment optimizes key parameters such as buffer composition, reaction temperature, time, and component concentration for the Cas13a reaction.

[0071] (a) Buffer optimization: First, the type and concentration of the buffer were optimized. Three commonly used buffer systems—HEPES, Tris-HCl, and phosphate buffer—were tested, with concentrations ranging from 10-50 mM and pH values ​​from 7.0-8.0. The results showed that Cas13a activity was highest and the fluorescence signal was strongest at 20 mM HEPES at pH 7.5.

[0072] Then, the divalent cations were optimized. Magnesium and manganese ions were tested in concentrations ranging from 2 to 20 mM. The results showed that 10 mM magnesium ions were the optimal condition; both excessively high and low concentrations reduced enzyme activity. Although manganese ions could also support the reaction, the background signal was high.

[0073] Next, the monovalent cations were optimized. Potassium and sodium ions were tested in concentrations ranging from 50 to 200 mM. The results showed that 100 mM potassium ions were the optimal condition, at which the hybridization efficiency of crRNA and miRNA and the cleavage activity of Cas13a were well balanced.

[0074] After comprehensive optimization, the optimal reaction buffer composition was determined to be: 20 mM HEPES pH 7.5, 10 mM MgCl2, and 100 mM KCl. This buffer was prepared as a 5× concentrate with the following composition: 100 mM HEPES pH 7.5, 50 mM MgCl2, and 500 mM KCl. It is stable for 6 months when stored at 4°C.

[0075] To verify the optimization effect, 1 fM miR-21 was detected using buffer solutions before and after optimization. The optimized fluorescence signal intensity increased by 3.5 times, the threshold time was advanced by 8 minutes, and the background signal was reduced by 40%, significantly improving the detection performance.

[0076] (II) Temperature Optimization: Four temperature points were tested: 25℃, 30℃, 37℃, and 42℃. The results showed that the reaction rate was fastest and the fluorescence signal was strongest at 37℃, the reaction was slow at 25℃, and although the initial reaction was fast at 42℃, the signal became unstable in the later stages. Therefore, 37℃ was chosen as the standard reaction temperature.

[0077] The reaction time was then optimized. Fluorescence signal changes were monitored at 37°C. For 10 fM miR-21, the fluorescence signal began to rise significantly at 15 minutes and reached a plateau at 25 minutes. To ensure the reliability of the detection and compatibility with samples of different concentrations, the standard reaction time was set to 30 minutes, which can be shortened to 20 minutes for high-concentration samples.

[0078] (III) Optimization of component concentration: Cas13a protein was tested at five concentrations: 10, 25, 50, 100, and 200 nM. The results showed that 50 nM provided the best cost-effectiveness; further increases in concentration offered limited signal enhancement but increased cost. Signal strength was significantly reduced below 50 nM.

[0079] Optimize crRNA concentration. Tests were conducted at 25, 50, 100, and 200 nM. Results showed that 50 nM yielded the best results, and the reaction was most efficient when maintained at a 1:1 molar ratio with Cas13a.

[0080] Optimize the report molecule concentration. Tests were conducted at 5, 10, 20, and 50 nM. Results showed that the signal-to-noise ratio was optimal at 10 nM; excessively high concentrations increased background signal, while excessively low concentrations limited signal strength.

[0081] Optimize the sample addition volume. In a 20 μL total reaction system, tests were conducted with 1, 2, 5, and 10 μL of sample. The results showed that a 5 μL sample volume provided sufficient detection sensitivity without diluting the reaction system and affecting efficiency, while also conserving sample.

[0082] After comprehensive optimization, the standard reaction system was determined to be a total volume of 20 μL, containing 50 nM Cas13a, 50 nM crRNA, 10 nM fluorescent reporter molecule, 1× reaction buffer, 5 μL sample, and nuclease-free water to a final volume of 20 μL. The mixture was then incubated at 37°C for 30 minutes.

[0083] Example 3: Detection Performance Evaluation (a) System evaluation of the detection sensitivity and linear range of the method of the present invention

[0084] A series of miR-21 synthetic standards were prepared, ranging from 0.01 fM to 1000 pM, including 17 concentration points: 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 500, 1000 fM and 1, 5, 10, 50, 100, 500, 1000 pM.

[0085] Three technical replicates were set up for each concentration point, and the detection was carried out according to the standard reaction conditions determined in Example 5. The changes in fluorescence signal were monitored in real time, and the threshold time Tt of each sample was recorded.

[0086] The results showed that, within the range of 0.1 fM to 100 pM, log 10 The concentration and Tt value showed a good linear relationship, and the linear regression equation was: Tt = -3.52 × log 10[miR-21]+18.75, correlation coefficient R²=0.998, coefficient of variation (CV) at each concentration point <5%.

[0087] Using a series of synthetic miR-21 standards at concentrations ranging from 0.1 fM to 100 pM, a linear relationship was established between the fluorescence threshold time and the logarithm of the miRNA concentration. Figure 2 The standard curve shown has a linear fitting coefficient R² = 0.998, proving that the method has good linearity and quantitative ability; the detection data of miR-21 standards at different concentrations are shown in Table 1 below.

[0088] Table 1: Detection results of miR-21 standards at different concentrations (n=3) miR-21 concentration Average Tt value (minutes) Standard deviation Coefficient of variation (CV) (%) 0.1 fM 29.2 0.68 2.3 1 fM 25.8 0.62 2.4 10 fM 22.3 0.51 2.3 100 fM 18.7 0.45 2.4 1 pM 15.2 0.38 2.5 10 pM 11.8 0.32 2.7 100 pM 8.5 0.28 3.3 For a concentration of 0.1 fM, the Tt values ​​for the three replicates were 29.2, 28.8, and 29.5 minutes, respectively. The fluorescence signal was significantly higher than that of the blank control, and the signal-to-noise ratio was >5, demonstrating that the detection limit reached 0.1 fM. Calculations show that adding 5 μL of 0.1 fM sample to a 20 μL reaction system is equivalent to approximately 60 miRNA molecules, approaching the single-molecule detection level.

[0089] At a concentration of 0.05 fM, the signal was partially repeated, but the repeatability was poor. Therefore, 0.1 fM was determined as the reliable detection limit.

[0090] For high-concentration samples, when the miR-21 concentration exceeds 100 pM, the fluorescence signal saturates within 5 minutes. Although it is still detectable, the quantitative accuracy decreases. For high-concentration samples that are outside the linear range, they can be diluted before detection.

[0091] In summary, the detection sensitivity of the method of the present invention is 0.1 fM, and the linear detection range spans 6 orders of magnitude, from 0.1 fM to 100 pM, meeting the detection needs of the vast majority of clinical samples.

[0092] (ii) Specificity verification experiments were conducted to verify the specific recognition capability of the method of the present invention for miRNA sequences. Several members of the let-7 family were selected for testing. These miRNAs have highly similar sequences, differing by only 1-2 bases. The mature sequences are as follows: let-7a: 5'-UGAGGUAGUAGGUUGUAUAGUU-3', SEQ ID NO.1 let-7b: 5'-UGAGGUAGUAGGUUGUGUGGUU-3', SEQ ID NO.2 let-7c: 5'-UGAGGUAGUAGGUUGUAUGGUU-3', SEQ ID NO.3 let-7d: 5'-AGAGGUAGUAGGUUGCAUAGUU-3', SEQ ID NO.4 We designed a specific crRNA targeting let-7a and detected let-7a, let-7b, let-7c, let-7d and unrelated miR-21 at a concentration of 10 pM.

[0093] Figure 3 The figure shows the experimental results of the specific detection of single-base differentially expressed miRNAs using the method of the present invention; Using a specific crRNA designed for let-7a as a detection probe, let-7a (the target) and its family members let-7b, let-7c (each differing from let-7a by one base), let-7d (distinguishing from let-7a by multiple bases), as well as the non-specific target miR-21 and a blank control without template, were simultaneously detected at a detection concentration of 10 pM. The results showed that let-7a produced the earliest fluorescence response (TtTt=12.3 min), while let-7b (TtTt=15.8 min) and let-7c (TtTt=16.2 min) had threshold time lags of 3.5 min and 3.9 min, respectively, and let-7d (TtTt=18.5 min) had a lag of 6.2 min. miR-21 and the blank control showed no effective fluorescence signal throughout the detection process. The detection curves clearly separated at the threshold line, demonstrating that the method of this invention can effectively distinguish miRNA sequences with single-base differences and exhibits high sequence specificity.

[0094] The results showed that let-7a produced a strong fluorescence signal with a Tt value of 12.3 minutes; let-7b had a Tt value of 15.8 minutes and a ΔTt value of 3.5; let-7c had a Tt value of 16.2 minutes and a ΔTt value of 3.9; let-7d had a Tt value of 18.5 minutes and a ΔTt value of 6.2; and miR-21 showed no obvious fluorescence signal within 30 minutes, which was comparable to the blank control.

[0095] Further testing was conducted to examine the effects of single-base mutations. Variant RNAs with the same miR-21 sequence but containing single-base mutations at different positions, namely positions 5, 10, 15, and 20, were synthesized. These variants were detected using miR-21 crRNA.

[0096] The results showed that the Tt values ​​of all single-base mutant variants were significantly delayed, with ΔTt ranging from 2.8 to 4.5 and an average ΔTt of 3.4, demonstrating that this method can effectively distinguish single-base differences. The effect was more significant when the mutation location was close to the center of the crRNA binding region.

[0097] The impact of mismatched base types was also tested. Three types of mismatches, AC, AG, and AA, were introduced at position 10 of miR-21. The results showed that the AC mismatch had the greatest impact, ΔTt=4.2; the AG mismatch was second, ΔTt=3.6; and the AA mismatch had a relatively small but still significant impact, ΔTt=2.9.

[0098] In summary, the method of the present invention has high sequence specificity, can effectively distinguish miRNAs with single-base differences, avoid cross-reaction, and is particularly suitable for detecting miRNA family members with similar sequences.

[0099] Example 4: Applicability verification for different sample types This embodiment verifies the applicability of the method of the present invention to different types of biological samples.

[0100] Group 1: Plasma Samples. 5 mL of peripheral blood was collected from healthy volunteers, anticoagulated with EDTA, and centrifuged at 3000 rpm for 10 minutes to separate the plasma. 5 μL of plasma was directly added to the Cas13a reaction system to detect endogenous miR-16.

[0101] First, the fluorescence curves obtained by detecting plasma samples, total RNA extracted from the same plasma sample, and synthesized miR-16 standards using the method of this invention are as follows: Figure 4 As shown, the results indicate that the Tt values ​​of the three samples are similar. Plasma samples without RNA extraction can be used directly for detection, producing a clear fluorescence signal curve with a Tt value of 15.8 minutes. As a control, total RNA was extracted from the same plasma sample using the Trizol method, and the RNA equivalent to 5 μL of plasma was used for detection, with a Tt value of 15.3 minutes. The difference between the two is only 0.5 minutes, a deviation of 3.2%, demonstrating the feasibility of direct plasma detection.

[0102] Calculations using a standard curve showed that the miR-16 concentration in this plasma sample was approximately 850 fM, consistent with reported miR-16 levels in normal human plasma.

[0103] Experiments have shown that the method of this invention can be directly used for plasma sample testing without the need for complex RNA extraction steps.

[0104] Group 2: Serum samples. Blood was left to stand at room temperature for 1 hour without anticoagulant to coagulate, and then centrifuged at 3000 rpm for 10 minutes to separate the serum. Direct detection of miR-21 in the serum yielded reliable results, with Tt values ​​close to those of homologous plasma samples, showing a difference of <5%.

[0105] Group 3: Urine samples. Midstream urine was collected, centrifuged at 12,000 rpm for 5 minutes to remove cells and impurities, and 5 μL of the supernatant was used for direct detection of miR-200 family members. Signals were successfully detected, but the sensitivity was slightly lower than that of plasma samples, possibly due to the lower concentration of miRNAs in urine.

[0106] Group 4: Tissue samples. Mouse liver tissue was collected, quick-frozen and ground in liquid nitrogen, and total RNA was extracted using the Trizol method. 100 ng of total RNA was used for detection, and miR-122, which is highly expressed in the liver, was successfully detected with high signal intensity and a Tt value of 8.2 minutes.

[0107] Group 5: Cell samples. The human hepatocellular carcinoma cell line HepG2 was cultured, and 1×10⁶ cells were collected. 5 Total RNA was extracted from cells, and 100 ng was used for detection. The high expression of miR-21 and miR-155 in cancer cells was successfully detected.

[0108] Group 6: Exosome samples. Exosomes were enriched from cell culture supernatant by ultracentrifugation, and 5 μL was directly taken after lysis for detection. miR-21 in exosomes was successfully detected, although the concentration was low, the signal was clear.

[0109] Group 7: Single-cell samples. A single HepG2 cell was picked up using a micropipette and placed in a tube containing 5 μL of lysis buffer. Lysis was performed at 37°C for 5 minutes, followed by direct addition to the Cas13a reaction system. See [link to results] for single-cell miRNA detection experiments. Figure 5 Using the method of this invention to detect miR-21 in a single HepG2 liver cancer cell, a clear fluorescence signal was successfully obtained, and miR-21 at the single-cell level was successfully detected. The Tt value was 22.5 minutes. The single-cell miR-21 copy number was calculated to be approximately 1200 using a standard curve, which is consistent with the literature reports, proving that the method is suitable for single-cell analysis.

[0110] In summary, the method of the present invention has broad sample applicability and can be used for various sample types such as plasma, serum, urine, tissue, cells, exosomes, and single cells. For body fluid samples, it can achieve direct detection without RNA extraction, which greatly simplifies the operation process.

[0111] Example 5: Comparison and validation with the gold standard qRT-PCR method This embodiment compares and verifies the method of the present invention with the current gold standard stem-loop qRT-PCR method.

[0112] Plasma samples from 20 clinical liver cancer patients were collected, and miR-21 expression levels were detected using the Cas13a method of this invention and the commercially available TaqMan stem-loop qRT-PCR kit.

[0113] For the CRISPR-Cas13a trans-cleavage method, 5 μL of plasma was used for direct detection, with three technical replicates per sample. For the qRT-PCR method, total RNA was extracted from 200 μL of plasma according to the manufacturer's instructions, followed by tailing and reverse transcription, and then real-time quantitative PCR was performed, with three technical replicates per sample.

[0114] Correlation analysis was performed on the detection results of the two methods: (See results below) Figure 6 The results showed that the relative expression levels of miR-21 in the 20 samples were highly positively correlated between the two methods, with a Pearson correlation coefficient r = 0.96 and p < 0.0001. Linear regression analysis showed that the slope was close to 1 and the intercept was close to 0, proving that the quantitative results of the two methods were highly consistent.

[0115] For a single sample, the relative deviation between the two methods was between -8.5% and +6.8%, with an average absolute deviation of 4.2%, which meets the requirements for clinical testing.

[0116] The performance comparison of the miRNA detection method of the present invention with existing stem-loop qRT-PCR and tailing qRT-PCR methods is shown in Table 2 below.

[0117] Table 2: Performance comparison between the miRNA detection method of the present invention and existing miRNA detection methods Performance indicators The Cas13a method of the present invention stem loop method qRT-PCR Tailing method qRT-PCR Is reverse transcription necessary? no yes yes Is PCR amplification necessary? no yes yes Detection time 20-40 minutes 3-4 hours 2.5-3.5 hours Detection sensitivity 0.1 fM 1 fM 0.5 fM Linear range 6-7 orders of magnitude 7 orders of magnitude 6 orders of magnitude Sample quantity 5 μL plasma 200 μL plasma 100 μL plasma Single base differentiation ability Excellent (ΔTt≥3) good medium Operational complexity Simple (one-step reaction) Complex (multi-step operation) Medium (two steps in one) Automation level Easy to automate Automatable Automatable Cost per test About 20 yuan Approximately 50 yuan Approximately 35 yuan As shown in Table 1 above, in terms of operation time, the qRT-PCR method requires RNA extraction, reverse transcription, and PCR amplification, totaling about 3.5 hours; the Cas13a method of this invention does not require RNA extraction, reverse transcription, and PCR amplification, but only requires direct mixing and incubation at 37°C, totaling about 40 minutes, which shortens the time by about 80%.

[0118] Regarding sample volume, stem-loop qRT-PCR requires 200 μL of plasma for RNA extraction, and tailing qRT-PCR requires 100 μL of plasma. The method of this invention only requires 5 μL, reducing sample volume by 97.5%, making it particularly suitable for precious clinical samples.

[0119] In terms of operational complexity, qRT-PCR requires three independent steps: RNA extraction, reverse transcription, and PCR, each of which requires accurate pipetting and temperature control; the method of this invention only requires one sample mixing and isothermal incubation, making it simple to operate.

[0120] In terms of cost, based on reagent consumption, the cost per test of the method of this invention is significantly lower than the cost per test of qRT-PCR.

[0121] In summary, the quantitative results of the method of this invention are highly consistent with the gold standard qRT-PCR method, and the accuracy has been fully verified. At the same time, it has significant advantages in terms of detection speed, sample volume, ease of operation and cost.

[0122] Example 6: Detection of plasma miRNAs in clinical liver cancer patients This embodiment applies the method of the present invention to a diagnostic study of clinical liver cancer patients.

[0123] Plasma samples were collected from 40 patients with liver cancer and 30 healthy controls. All liver cancer patients were pathologically diagnosed, including 20 early-stage patients and 20 late-stage patients. Healthy controls underwent physical examinations to rule out tumors and serious diseases. The study was approved by the hospital's ethics committee, and all participants signed informed consent forms.

[0124] The method of this invention was used to detect three known liver cancer-associated miRNAs, miR-21, miR-155, and miR-10b, in plasma. Five μL of plasma was collected from each sample, and the three miRNAs were detected separately, with three technical replicates for each indicator.

[0125] The results showed that the average concentration of miR-21 in the plasma of liver cancer patients was 2350 fM, significantly higher than 680 fM in the healthy control group, with a statistically significant difference. miR-155 was 1580 fM in the patient group and 520 fM in the control group. miR-10b was 890 fM in the patient group and 180 fM in the control group. All three miRNAs were significantly elevated in the patient group.

[0126] Further analysis revealed that while the levels of three miRNAs in early-stage liver cancer patients were lower than those in late-stage patients, they were still significantly higher than those in healthy controls, demonstrating that these miRNAs can serve as early diagnostic biomarkers.

[0127] ROC curve analysis was performed to assess diagnostic efficacy. The AUC of miR-21 was 0.89, with a sensitivity of 85% and a specificity of 83% at a cutoff value of 1200 fM. The AUC of miR-155 was 0.86. The AUC of miR-10b was 0.91.

[0128] A combined diagnostic model of three miRNAs was established. Through logistic regression analysis, the AUC of the combined model was improved to 0.94, with a sensitivity of 90% and a specificity of 87%, which were significantly better than those of single indicators. The diagnostic accuracy can be improved by combining multiple indicators.

[0129] This embodiment demonstrates that the method of the present invention can be effectively applied to the detection of plasma miRNA in clinical cancer patients, has good diagnostic efficacy, and has application value in the early diagnosis and screening of tumors.

[0130] Example 7: Simultaneous detection of multiple miRNAs This embodiment develops a method for simultaneous detection of multiplex miRNAs based on multicolor fluorescence.

[0131] Three fluorescent reporter molecules were prepared by selecting three different combinations of fluorescent and quenching groups: Reporter molecule 1: 5'-FAM-UUUUU-BHQ1-3', SEQ ID NO. 5 Reporter molecule 2: 5'-HEX-UUUUU-BHQ1-3', SEQ ID NO. 6 Reporter molecule 3: 5'-ROX-UUUUU-BHQ2-3', SEQ ID NO.7 Three clinically relevant miRNAs were selected: miR-21, miR-155, and miR-16, and specific crRNAs were designed for each.

[0132] Add the following to the same reaction system: (1) Cas13a protein 50 nM (2) miR-21 crRNA 50 nM (3) miR-155 crRNA 50 nM (4) miR-16 crRNA 50 nM (5) FAM reporter molecule 10 nM (for miR-21 detection) (6) HEX reporter molecule 10 nM (for miR-155 detection) (7) ROX reporter molecule 10 nM (for miR-16 detection) (8) Reaction buffer and sample A real-time quantitative PCR instrument with multi-channel fluorescence detection function was used to simultaneously monitor the fluorescence signals of three channels: FAM, HEX, and ROX.

[0133] The system was first validated using a mixed standard containing known concentrations of three miRNAs. The validation results are as follows: Figure 7 As shown, the results indicate that the three fluorescence channels each generate specific signals without interfering with each other, and their respective standard curves exhibit good linearity with R² > 0.99, demonstrating that the method can achieve multiplex detection.

[0134] Then, clinical plasma samples were used for testing. Three miRNAs were simultaneously detected in a single reaction well, successfully obtaining three independent fluorescence curves, and the concentrations of the three miRNAs were quantified separately. The deviation was <8% compared to the results of individual detection.

[0135] Multiplex detection methods reduce the detection time for a single sample from 90 minutes to 30 minutes, and reduce reagent and sample consumption by 67%, significantly improving detection efficiency and cost-effectiveness.

[0136] This embodiment demonstrates that the method of the present invention can achieve simultaneous detection of multiple miRNAs through multicolor fluorescent labeling, making it suitable for high-throughput screening and expression profiling analysis.

[0137] Example 8: Study on reagent kit stability and storage conditions This embodiment systematically studies the stability and optimal storage conditions of each component of the kit.

[0138] Cas13a protein stability (tested using Cas13a protein solution): The purified Cas13a protein was aliquoted into solutions and stored at different temperatures: (1) -80℃: No significant change in activity after 18 months (2) -20℃: Activity remains >95% after 12 months (3) 4℃: The activity decreased to about 85% after 3 months. (4) Room temperature: Activity decreased to approximately 60% after 1 week. Freeze-thaw stability test: The same batch of Cas13a protein was repeatedly frozen and thawed, and the change in activity was detected. The results showed that the activity remained >90% within 3 freeze-thaw cycles, but decreased to about 80% after 5 freeze-thaw cycles. It is recommended to aliquot and store the protein to avoid repeated freeze-thaw cycles.

[0139] crRNA stability (tested with crRNA solution): (1) -80℃ or -20℃: No degradation after 12 months (2) 4℃: Slight degradation after 6 months, but activity remains >90%. (3) Room temperature: Significant degradation occurred after 1 month. Stability of fluorescently labeled RNA reporter molecules (tested using fluorescently labeled RNA reporter molecule solutions): Fluorescently labeled RNA reporter molecules are sensitive to light and must be stored away from light. (1) Stable for 12 months at -20℃ away from light. (2) Stable for 6 months at 4℃ away from light. (3) Room temperature, protected from light: fluorescence intensity decreased by about 15% after 1 month. (4) Room temperature light exposure: fluorescence intensity decreased by >30% after 1 week Lyophilized formulation development: To improve the stability and storage convenience of the kit, a lyophilized formulation was developed. Cas13a protein, crRNA, fluorescent reporter molecules, and reaction buffer were mixed, and then protective agents (trehalose and mannitol) were added before lyophilization to form a powder.

[0140] Stability testing of lyophilized formulations: (1) 4℃: After 12 months, the activity remains >95% upon reconstitution. (2) 25℃: After 6 months of reconstitution, the activity remains >90%. (3) 37℃: After 3 months of reconstitution, the activity remains >85%. Lyophilized formulations significantly improve the storage stability of reagent kits, simplify cold chain transportation requirements, and are particularly suitable for resource-constrained areas and long-term storage needs.

[0141] Based on the stability study results, the following storage conditions are recommended for the kit: (1) Liquid reagent kit: Store at -20℃, shelf life 12 months (2) Lyophilized reagent kit: Store at 4℃ for 12 months; store at -20℃ for 24 months. (3) Transportation conditions: cold chain transportation, dry ice or ice packs for insulation, transportation time not exceeding 48 hours. The present invention provides a quantitative fluorescence detection method and kit for miRNA based on CRISPR-Cas13a trans-cleavage, which can be used for tumor diagnosis, tumor subtyping, efficacy monitoring, prognostic assessment, and diagnosis of cardiovascular diseases. Applicable tumors include liver cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, breast cancer, prostate cancer, ovarian cancer, leukemia, or lymphoma.

[0142] This invention, through the above embodiments, details the technical principles, implementation schemes, and application effects of a CRISPR-Cas13a trans-cleavage-based method for quantitative detection of miRNA. This method successfully overcomes many limitations of existing technologies, achieving rapid, highly sensitive, and highly specific quantitative detection of miRNA without reverse transcription or PCR amplification, providing strong technical support for the application of miRNA in clinical diagnosis and scientific research.

[0143] The scope of protection of this invention is not limited to the specific embodiments described above. Any equivalent substitutions or obvious modifications based on the technical concept of this invention should be included within the scope of protection of this invention.

Claims

1. A quantitative fluorescence detection method for miRNA based on CRISPR-Cas13a trans-cleavage, characterized in that... Includes the following steps: The first step is to provide a sample containing the miRNA to be tested; The second step involves mixing the sample to be tested with the detection reagents to construct a reaction system. This reaction system includes Cas13a protein at a concentration of 20–200 nM, specific crRNA at a concentration of 20–200 nM, fluorescently labeled RNA reporter molecules at a concentration of 5–50 nM, and a reaction buffer. The specific crRNA contains a guide sequence complementary to the target miRNA, with a guide sequence length of 18–28 nucleotides, and is either completely complementary to the target miRNA or allows for mismatches of no more than 2 bases. The reaction buffer consists of 20 mM HEPES (pH 7.5), 10 mM MgCl2, and 100 mM KCl. The third step is isothermal incubation and real-time monitoring of fluorescence signals: The reaction system is incubated at a constant temperature of 30-42℃ for 15-60 minutes. During the incubation, the miRNA to be tested forms a double strand with the crRNA, which activates the trans-cleavage activity of Cas13a, cleaving the fluorescently labeled RNA reporter molecule and releasing a fluorescence signal. The instrument collects fluorescence signals every 30-120 seconds and records the time point or cycle number when the fluorescence signal reaches the set threshold. The fourth step is data analysis and quantitative calculation: a standard curve is established in advance using miRNA standards of a series of concentrations. The fluorescence signal collected during the isothermal incubation process is compared with the standard curve to calculate the concentration of the miRNA to be tested in the sample.

2. The detection method according to claim 1, characterized in that, The preferred conditions for the reaction system are: Cas13a protein at a concentration of 50 nM, specific crRNA at a concentration of 50 nM, and fluorescently labeled RNA reporter molecule at a concentration of 10 nM; the reaction system is incubated at a constant temperature of 37°C for 30 minutes.

3. The detection method according to claim 1, characterized in that, Multiple specific crRNAs targeting different miRNAs and RNA reporter molecules labeled with different fluorescent groups can be added to the same reaction system to achieve simultaneous detection of 2 to 6 miRNAs, with peak emission wavelength intervals of fluorescent groups ≥30 nm.

4. The detection method according to claim 1, characterized in that, The miRNA to be tested is selected from one or more of the following families: miR-21, miR-16, miR-155, miR-210, miR-122, let-7 family, and miR-200 family.

5. The detection method according to claim 1, characterized in that, The fluorescently labeled RNA reporter molecule has a structure of 5'-fluorescent group-ribonucleotide sequence-quencher group-3', and its ribonucleotide sequence consists of 4 to 10 uracil nucleotides, preferably 5 uracil nucleotides.

6. The detection method according to any one of claims 1 to 5, characterized in that, Its detection performance indicators are as follows: detection sensitivity is 0.1 fM, linear detection range is 0.1 fM to 100 pM; the specific crRNA is complementary to the bases of the miRNA to be tested, and can effectively distinguish miRNAs with single-base differences.

7. The detection method according to any one of claims 1 to 4, characterized in that, The sample can be a purified RNA solution after RNA extraction, or a sample that has undergone simple lysis treatment, including but not limited to blood, plasma, serum, urine, saliva, cerebrospinal fluid, tissue homogenate, cell lysate, exosome solution, and single-cell lysate.

8. A miRNA fluorescence quantitative detection kit, characterized in that, The following reagents are included: (1) Cas13a protein; (2) One or more specific crRNAs, said specific crRNAs containing a guide sequence complementary to the miRNA to be tested; (3) One or more fluorescently labeled RNA reporter molecules, wherein the structure of the fluorescently labeled RNA reporter molecule is 5'-fluorescent group-ribonucleotide sequence-quencher group-3', wherein the ribonucleotide sequence is composed of 4 to 10 uracils; multiple fluorescently labeled RNA reporter molecules designed for the simultaneous detection of multiple target miRNAs are RNA reporter molecules labeled with different fluorescent groups; (4) The reaction buffer consists of: 20 mM HEPES pH 7.5, 10 mM MgCl2, and 100 mM KCl; The miRNA to be tested is preferably selected from miR-21, miR-16, miR-155, miR-210, miR-122, let-7 family, and miR-200 family.

9. The detection kit according to claim 8, characterized in that, The Cas13a protein, specific crRNA, fluorescently labeled RNA reporter molecule, and reaction buffer are all individually packaged solutions; or the Cas13a protein, specific crRNA, fluorescently labeled RNA reporter molecule, and reaction buffer are mixed, and then trehalose and mannitol are added as protective agents, followed by freeze-drying to produce a powder.

10. The detection kit according to claim 8 or 9, characterized in that, Also includes: Positive control miRNA standard, negative control solution, RNase inhibitor, nuclease-free water, miRNA standard of various concentrations, optically clear reaction tubes or reaction plates.

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