Method for miRNA one-pot detection by activating Cas12a through transcription amplification of RT-RPA integrated scaffold crRNA

Abstract

The invention provides a biosensor for detecting miRNA by a one-pot method based on transcription amplification activation of Cas12a trans-cleavage of RT-RPA and scaffold crRNA, and belongs to the technical field of biological analysis. According to the invention, miRNA is amplified into double-stranded DNA with a T7 promoter by using RT-RPA (reverse transcription recombinase polymerase amplification). And a transcription product is combined with a 5-terminal extension sequence of a split T7 promoter with a scaffold crRNA transcription template to form a three-way connector. After the scaffold crRNA is transcribed, one part of the scaffold crRNA is combined with the cohesive end of a transcription template to form a DNA-RNA hybrid complex, and the complex is used as a spacer crRNA and an activator and is assembled with the scaffold crRNA to perform a complete crRNA function and activate Cas12a trans-cleavage activity. And the reaction is carried out in one tube, so that cross contamination is avoided. By utilizing the biosensor provided by the invention, the content of miRNA in cancer cells and serum samples can be effectively detected. Due to simplicity, high sensitivity and good adaptability, the method becomes a real-time detection method with great potential in the field of molecular diagnosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioanalysis, and specifically relates to the preparation and application of a RT-RPA integrated scaffold crRNA transcription amplification activated Cas12a for one-pot detection of miRNA biosensor. Background Art

[0002] MicroRNAs (miRNAs) are a class of small noncoding RNA molecules that play key roles in regulating gene expression and have been widely documented to be involved in developmental processes and disease pathogenesis. Of particular note, miRNAs are significantly overexpressed in cancer tissues compared to normal tissues, highlighting their potential as cancer biomarkers. In addition, these molecules play key roles in cancer proliferation, migration, drug resistance, and immune regulation in the tumor microenvironment, further emphasizing their importance in tumor formation and cancer progression. Therefore, accurate detection of miRNAs is essential for understanding their regulatory mechanisms and their potential as biomarkers for disease diagnosis and prognosis. Due to the short sequences of miRNAs and the high homology between family members, the highly sensitive detection of miRNAs faces major challenges. Traditional methods for detecting miRNAs mainly include quantitative reverse transcription polymerase chain reaction (qRT-PCR), second-generation sequencing, and Northern blotting. However, the use of these technologies requires expensive equipment and reagents, skilled technicians, and complex sample preparation and processing procedures, which limit their practical application and development. Therefore, it becomes crucial to develop a convenient, low-cost, and ultrasensitive miRNA detection strategy.

[0003] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) systems, especially those based on CRISPR-Cas12a, have made significant progress in the field of molecular detection. However, CRISPR systems have limitations in detection sensitivity, target range, and activation mechanism. In order to improve the sensitivity of detection, CRISPR systems are often used in combination with various nucleic acid amplification technology (NAAT) methods, such as exponential isothermal amplification, loop-mediated isothermal amplification, and polymerase chain reaction, which have been reported in the past few years. Although these methods can improve sensitivity, they usually use CRISPR systems and NAAT separately instead of integrating them into a test tube reaction. Such multi-step detection is not only cumbersome, but also increases the time of the entire detection process, and cannot meet the needs of environments that require rapid diagnosis. In addition, most CRISPR systems rely on exogenously added activators to activate Cas12a protein activity, which requires additional steps and strict concentration control to ensure effective CRISPR activation, which may increase the risk of nonspecific activation of Cas12a protein even in the absence of target DNA, thereby leading to off-target effects of the system. Therefore, there is an urgent need to develop new Cas12a-based diagnostic tools to address these related issues.

[0004] Split crRNA has emerged as an innovative method in the CRISPR-Cas12a system, enhancing the detection capabilities of molecular diagnostics. This method involves dividing crRNA into two different parts: a scaffold crRNA with a conserved sequence and a spacer RNA with a variable sequence. Recent studies have shown that these two parts can be reassembled with the Cas12a protein to form a ribonucleoprotein (RNP) complex with the same cutting ability as the wild-type Cas12a RNP. The Split crRNA system has been shown to be highly sensitive and specific for detecting miRNA and non-coding RNA, but the target RNA will form a secondary structure that will interfere with it. Studies have shown that after removing the scaffold sequence, Cas12a can also target the cutting of target DNA in vitro through a single crRNA spacer sequence. However, the experiment requires a higher concentration of crRNA, which may increase the risk of nonspecific binding and off-target effects. Therefore, it is very necessary to construct a new, convenient and sensitive split crRNA combined with Cas12a biosensor to detect miRNA. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention proposes a one-pot RT-RPA biosensor integrating scaffold crRNA transcription amplification and activation of Cas12a for miRNA detection, which has the advantages of rapidity, high sensitivity, high specificity, visualization and low equipment requirements.

[0006] The technical solution of the present invention is as follows:

[0007] 1. A RT-RPA-assisted scaffold crRNA transcription amplification activated Cas12a trans-cleavage for one-pot detection of miRNA biosensor, which includes three parts: RT-RPA amplification, scaffold crRNA transcription amplification and CRISPR system:

[0008] (1) RT-RPA amplification includes: a stem-loop probe that specifically recognizes microRNA, 10×RT Mix, HiScriptⅡ enzyme mixture, ERA basic, RPA upstream primer with T7 promoter, and RPA downstream primer;

[0009] (2) Scaffold crRNA transcription amplification includes: split T7-1 (comprising the scaffold crRNA sequence and the complementary sequence of the T7 promoter and a complementary sequence capable of binding to half of the target RNA region), split T7-2 (comprising a partial sequence of the T7 promoter (4 nt) and a sequence capable of binding to the other half of the target RNA region), split T7-3 (comprising a partial sequence of the T7 promoter (16 nt)), T7 RNA polymerase and NTPs;

[0010] (3) CRISPR system: Cas12a protein and reporter probe FQ.

[0011] 2. Connect miR-155 and the stem-loop probe and reverse transcribe cDNA. Amplify the double-stranded cDNA with T7 promoter through RPA. Then transcribe the target RNA under the action of T7 RNA polymerase. The target RNA is connected with split T7-1, split T7-2, and split T7-3 to form a three-way connector and a double-stranded T7 promoter with a gap site;

[0012] 3. The three-way connector described in step 1 carries a scaffold crRNA transcription template. After transcription, the scaffold crRNA binds to the sticky end of the DNA transcription template on split T7-1 to form a DNA-RNA hybrid complex. The DNA-RNA hybrid complex and the scaffold crRNA are assembled together to form a function similar to split crRNA, activating the Cas12a protein to cut the FQ reporter and generate a fluorescent signal. The complex formed by DNA-RNA acts as an activator and spacer crRNA in this system;

[0013] 4. Preferably, the 21nt-long RNA sequence acts as both scaffold crRNA and spacer crRNA;

[0014] 5. Preferably, the biosensor is used for ultrasensitive and efficient detection of miR-155 in one pot;

[0015] 6. Preferably, the biosensor is used for diagnosis of cancer cells and serum samples.

[0016] Compared with the prior art, the one-pot RT-RPA integrated scaffold crRNA transcription amplification activated Cas12a trans-cleavage biosensor for miRNA detection described in the present invention has the following advantages:

[0017] In this invention, we used a conserved sequence of 21 nucleotides, which serves as both scaffold crRNA and spacer crRNA, and can function as split crRNA. The DNA transcription template of the scaffold RNA can serve as an activation chain, and their combined action can activate the trans-cleavage activity of Cas12a. By transcribing the split crRNA, a portion of it binds to the sticky end of the transcription template to form a DNA-RNA hybrid. The scaffold crRNA and DNA-RNA hybrid complex activate the trans-cleavage activity of Cas12a. Based on this discovery, we constructed a new biosensor that integrates reverse transcription recombinase polymerase amplification with the CRISPR-Cas12a system to achieve ultra-sensitive one-pot detection of miRNA. This integrated detection significantly improves the sensitivity of the detection while simplifying the procedural steps, making the detection method faster and simpler. We first reverse transcribe the miRNA into cDNA, and then amplify the cDNA into double-stranded DNA with a T7 promoter by a recombinase polymerase. The double-stranded DNA is transcribed by T7 to produce RNA targets. The target is then linked to the split T7 promoter extension sequence with the scaffold crRNA transcription template to form a three-way connector, triggering the transcription of the scaffold crRNA, which then hybridizes with it to form a DNA-RNA complex. The scaffold crRNA and the complex jointly activate the trans-cleavage activity of Cas12a, resulting in an enhanced fluorescence signal. The detection limit of the biosensor is as low as 2.66aM. More importantly, we have successfully applied this detection method to clinical samples, including serum and cell samples, thus verifying its potential in practical biomedical research and clinical diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to further enhance the understanding of the present invention, the following is a brief introduction to the drawings required for the specific implementation method. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention. In the drawings:

[0019] Figure 1 A in the figure is a schematic diagram of the principle of Cas12a trans-cleavage activated by scaffold crRNA transcription amplification; B is a schematic diagram of the principle of a biosensor for miRNA detection based on one-pot RT-RPA assisted scaffold crRNA transcription amplification activation Cas12a trans-cleavage;

[0020] Figure 2 A in the middle is a feasibility diagram of scaffold crRNA transcription amplification activation Cas12a trans-cleavage of single-stranded DNA and plasmid conformational changes;

[0021] Figure 3 To verify the electrophoresis diagram of the binding of the transcribed crRNA to its template;

[0022] Figure 4 This is a diagram for optimizing the length of scaffold crRNA of the present invention;

[0023] Figure 5 Optimization diagram for magnesium ion concentration and reaction temperature;

[0024] Figure 6 Optimization diagram for Cas12a and reporter probe FQ concentration;

[0025] Figure 7 This is a feasibility diagram for one-pot detection of miR-155;

[0026] Figure 8 One-pot method for miRNA detection in cells and clinical samples;

[0027] Fig. 9 To compare the RT-qPCR detection of miRNA-155 extracted from HepG2, MCF-7, HeLa, and MCF-10A cell lines with this method in biosensing applications;

[0028] Fig.10 To test the practical application of detecting miRNA-155 in cell lysate. DETAILED DESCRIPTION

[0029] Sequence Listing:

[0030]

[0031]

[0032] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0033] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the disclosure and materials cited therein are hereby incorporated by reference.

[0034] Technical equivalents to the specific embodiments described that are apparent to those skilled in the art using no more than routine experimentation are intended to be encompassed by this application.

[0035] The experimental methods in the following examples are all conventional methods unless otherwise specified. The instruments and equipment used in the following examples are all conventional laboratory instruments and equipment unless otherwise specified; the experimental materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0036] Example 1: Scaffold crRNA transcription amplification activates Cas12a trans-cleavage principle

[0037] like Figure 1 As shown in A, the hairpin-shaped DNA transcription template carries the T7 first promoter, the T7 second promoter, and the scaffold crRNA template. T7 RNA polymerase is added to start transcribing the scaffold crRNA. The transcribed scaffold crRNA will bind to the circular DNA transcription template to form a DNA-RNA hybrid complex. This complex acts as a spacer crRNA and a single-stranded activator. This complex and the scaffold crRNA work together to activate the trans-cutting activity of Cas12a. The reporter probe FQ is then cut, which is extended by 10 bases at the FAM fluorescent group end and 11 bases at the quenching group end. These 21 bases are complementary to the scaffold crRNA sequence, and there are 5 bases (TTATT) between the two groups to form a bulge. The activated Cas12a will cut the five protruding bases, and then the scaffold crRNA will fall off, bind to the DNA template, and participate in the cycle.

[0038] Example 2: RT-RPA integrated scaffold crRNA transcription amplification activated Cas12a trans-cleavage for one-pot miRNA detection

[0039] like Figure 1 As shown in B, based on the principle of Example 1, miRNA is reverse transcribed into cDNA, and then cDNA is amplified into double-stranded DNA with T7 promoter by recombinase polymerase. Double-stranded DNA is transcribed by T7 to produce RNA target. After that, the target is connected to the 5-terminal extension sequence of the split T7 promoter with the scaffold crRNA transcription template to form a three-way connector, triggering the transcription of scaffold crRNA, and then hybridizing with it to form a DNA-RNA complex. The scaffold crRNA and DNA-RNA complex jointly activate the trans-cleavage activity of Cas12a, resulting in enhanced fluorescence signal.

[0040] Example 3: Steps involved in the implementation of the present invention

[0041] (1) In vitro transcription:

[0042] 20 μL transcription reaction system transcribes scaffold crRNA, transcription template (0.5 μL, 2 μM), second T7 promoter sequence (0.5 μL, 2 μM), AmpliScribe T7 reaction buffer (2 μL, 10×), 2 μL 10 mm DTT, RNase inhibitor (0.5 μL, 20 U), T7 RNA polymerase (2 μL, 12 U), RNase (6 μL, 10 mM) and 6.5 μL RNase-free water. Transcribe at 37°C for 2 h, then treat part of the reaction mixture with DNase I (50 U / mL) for 30 min, and then put it at 95°C for 5 min to inactivate DNase I.

[0043] (2) Gel electrophoresis analysis:

[0044] Utilize 10% non-denaturing polyacrylamide gel electrophoresis (PAGE) to analyze different samples. 10 μ L sample and loading buffer (1.1 μ L 10X) are added to the wells, and 10% non-denaturing polyacrylamide gel electrophoresis analysis is performed in 1X TBE buffer (45mM Tris, 45mM boric acid, 10mM EDTA, pH=8.0), at room temperature, constant voltage 110V, 60min. Plasmid samples are analyzed using 1% agarose gel electrophoresis in 1X TBE buffer. Electrophoresis is performed at a constant voltage of 100V for 90min, and the sample load of each channel is 10 μ L. The gel is then washed three times and stained with ethidium bromide reagent (diluted with 1X TBE). The gel is imaged and analyzed using a 365nm ultraviolet gel imaging operating system.

[0045] (3) Spectral measurement and analysis

[0046] The reaction was carried out in 1×NEBuffer 2.1 (0.2mM EDTA, 9mM Tris-HCl, 9mM boric acid, pH 7.9), and the mixture contained 1μL LbCas12a (1μM), 1μL transcription mixture (2μM) and 1μL FQ reporter gene (5μM). The total reaction volume was 20μL. After the reaction was completed, 80μL of water was added to quantify the fluorescence intensity. The emission slit and the excitation slit were both set at 5.0nm. Using an F-7000 spectrophotometer, the excitation wavelength was 480nm, and the emission spectrum from 500nm to 600nm was recorded.

[0047] (4) Reverse transcriptase recombinase polymerase amplification

[0048] Under the catalysis of reverse transcriptase, miRNA is converted into cDNA by adding a stem loop at the 3' end. The reverse transcription system consists of miRNA, 10×RT Mix and Hiscript II Enzyme Mix. Add 1μL stem loop primer (2μM), 2μL 10×RT Mix, 2μL HiScriptⅡEnzyme Mix and 5μL RNase-free ddH 2 O. The reaction process was 25℃, 5min; 42℃, 15min. The target cDNA was specifically amplified by recombinase polymerase amplification. RPA was performed by GenDxERAKit. A 25μL RPA reaction included 5μL ERA basic, 10μL reaction buffer, 5μL cDNA template, 1.25μL RPA front primer (10nM), 1.25μL RPA rear primer (10nM) and 2.5μL water. The specific operation was referred to the instruction manual of the kit. The mixed system was incubated at 37℃ for 20min to obtain a large amount of specific amplification products.

[0049] (5) RT-qPCR analysis

[0050] Total RNA was extracted from MCF-7, HeLa, HepG2, MCF-10A cells and serum using the UNIQ-10 column Trizol Total RNA Purification Kit (SK1321). cDNA samples were prepared using the miRNA First Strand cDNA Synthesis Kit. The total volume of 20 μL system contained 2 μL total miRNA, 2 μL miRNA RT enzyme mix, 10 μL 2× miRNA RT reaction buffer and 6 μL RNA-free H 2 O incubation at 42°C for 60 min, then at 95°C for 3 min, and cDNA samples were stored at -20°C for later use. RT-qPCR analysis was performed using the manufacturer's instructions on an ABI StepOnePlus qPCR instrument using SybrGreen PCR Master Mix. The 20 μL reaction mixture consisted of 2 μL cDNA, 10 μL SybrGreen qPCR Master Mix, 0.4 μL upstream primer (10 μM), 0.4 μL downstream primer (10 μM), and 7.2 μL nuclease-free water. The qPCR conditions were: 95°C for 3 min, followed by 40 cycles at 95°C for 15 s, 57°C for 20 s, and 72°C for 30 s. 2 -ΔΔCt The expression level of U6 gene was detected by the method, and the relative expression level of cell samples was analyzed.

[0051] Example 4: Verification of the feasibility of the principle of scaffold crRNA transcriptional amplification to activate Cas12a trans-cleavage

[0052] Gel preparation and spectra were the same as in Example 3; Figure 2 B Lane 1 is single-stranded DNA; Lane 2 shows the transcription mixture plus Cas12a, and it can be found that the single-stranded DNA is cut; Lane 3 is the DNA transcription template digested with DNase I, and it can be found that the single-stranded DNA has not changed; Lane 4 is based on Lane 3 and an additional DNA transcription template is added, and it is found that the single-stranded DNA is cut again. Figure 2 C Lanes 1 and 5 were used as negative controls. Two distinct bands were observed in lanes 1 and 5 for pUC57 and pET28a, indicating the presence of supercoiled and open circles in the intact plasmid. The top band represents the open circle, and the bottom band represents the supercoiled conformation. After adding transcription mix and Cas12a to the plasmid, we found that the banding pattern changed significantly when the target was present. We then linearized the plasmid with BamH1. We added BamH1 to the plasmid and observed the disappearance of the supercoiled and open circle bands, and a new band appeared in the middle ( Figure 2 C, lanes 3 and 7). This confirms that the plasmid undergoes a supercoiled-circular-opening-linear transition due to the enzymatic activity of Cas12a. Finally, we added the linearized plasmid to the transcription mixture together with Cas12a and observed no change in the bands ( Figure 2 C, lanes 4 and 8). This confirms that under normal circumstances, Cas12a does not participate in the cleavage of non-target double-stranded DNA. In addition, we also used full-length crRNA plus activator to compare with our method and found that the fluorescence intensity was similar ( Figure 2 D). Finally, we used gel electrophoresis to verify the generation of DNA-RNA hybrid complexes during transcription, such as Figure 3 The fourth lane.

[0053] Example 5: Optimization of scaffold crRNA length

[0054] Fluorescence measurement analysis is the same as in Example 3; we selected the scaffold crRNA corresponding to the LbCas12a protein to optimize the length, and found that the scaffold crRNA of 21 bases worked best. Finally, we used transcription templates of different concentrations to measure its fluorescence working curve ( Figure 4 ).

[0055] Example 6: Optimization of experimental conditions

[0056] (1) Optimization of magnesium ion concentration

[0057] Fluorescence measurement analysis was the same as in Example 3; except that the magnesium ion concentration was changed (5, 10, 20, 30 mM); the results are shown in Figure 5 A. The fluorescence signal is highest when the magnesium ion concentration is 20 mM.

[0058] (2) Reaction temperature optimization

[0059] Fluorescence measurement analysis was the same as in Example 3; reaction temperature (25, 30, 37, 42°C); results are shown in Figure 5 B. The fluorescence signal is highest when the reaction temperature is 37°C.

[0060] (3) Cas12a concentration optimization

[0061] Fluorescence measurement analysis is the same as in Example 3; Cas12a concentration (0, 5, 10, 25, 50 nM); results are shown in Figure 6 A. The fluorescence signal is highest when the concentration of Cas12a is 50nM.

[0062] (4) Optimization of reporter probe FQ concentration

[0063] Fluorescence measurement analysis was the same as in Example 3; reporter probe FQ concentrations (0, 50, 100, 150, 250 nM); results are shown in Figure 6 B. The fluorescence signal is highest when the concentration of reporter probe FQ is 250 nM.

[0064] Example 7: RT-RPA integrated scaffold crRNA transcription amplification activated Cas12a trans-cleavage for one-pot miRNA performance analysis

[0065] (1) Electrophoresis analysis

[0066] Results Figure 7 A, B; Same as Example 3; It was found that new bands appeared after adding reverse transcriptase and T7 RNA polymerase, indicating that the assembly was successful;

[0067] (2) Spectral analysis

[0068] Results Figure 7 C; Same as Example 3, signal amplification occurs only when there is a target and Cas12a;

[0069] (3) Establishment of standard curve

[0070] Results Figure 7 D, E; Same as Example 3, nonlinear and linear fitting were performed for the fluorescence intensity corresponding to different miR-155 concentrations, and the linear relationship between the FQ fluorescence intensity at 530nm and the miRNA-155 concentration was detected from 1fM to 10pM, and the regression equation was F = 336.08 × lgC-453.35 (R 2=0.9917);

[0071] (4) Specificity analysis

[0072] Results Figure 7 F: Same as Example 3, except that the target miRNA (let-7a, miR-21, miR-16, miR-27a) or miR-155 mutant was changed. The results showed that the sensor had good specificity.

[0073] Example 8: One-pot detection of miRNA in cells and clinical samples

[0074] (1) Detection of miR-155 in cells

[0075] The preparation method is the same as that of Example 3; the results are shown in Figure 8 ; The expression level of miRNA-155 extracted from HepG2, MCF-7, HeLa and MCF-10A cell lines was evaluated by RT-qPCR, and it was found that the miR-155 content was the highest in MCF-10A cells; The target miRNA-155 concentration estimated by this method and the traditional RT-qPCR method proved that our method can achieve the same results as RT-qPCR. The results are shown in Fig. 9 We used this method and RT-qPCR to compare the miR-155 content in four cell lines and found that MCF-10A cells were normal cells.

[0076] (2) Detection of miR-155 in serum

[0077] like Figure 8 In D, the expression levels of miRNA-155 in patients and healthy donors analyzed by this method (blue) and RT-qPCR (red), demonstrating that our method can distinguish between healthy and patients. Fig.10 , using RT-qPCR real-time fluorescence monitoring of different concentrations of miRNA-155 (10aM, 100aM, 1fM, 10fM and 100fM). Figure B shows that in the range of 10aM to 100fM, there is a good linear relationship between the Ct value and the logarithm of the miRNA-155 concentration, and the regression equation is F = -3.3786LgC + 33.287 (R 2 =0.9993).

[0078] Note: Serum specimens from breast cancer patients and normal subjects were obtained from the Hunan Metabolic Fatty Liver Disease Clinical Research Center, Clinical Research Institute, Nanhua Hospital Affiliated to Hengyang Medical College, University of South China, and approved by the Medical Ethics Committee of Nanhua Hospital Affiliated to University of South China (No. 2023-ky-163).

[0079] The preferred specific embodiments of the present invention are described in detail above. It should be understood that a person skilled in the art can make many modifications and changes based on the concept of the present invention without creative work. Therefore, any technical solution that can be obtained by a person skilled in the art through logical analysis, reasoning or limited experiments based on the concept of the present invention on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A RT-RPA integrated scaffold crRNA transcription amplification activated Cas12a trans-cleavage for one-pot detection of miRNA biosensor, characterized in that, The biosensor consists of three parts: RT-RPA amplification, scaffold crRNA transcription amplification and CRISPR system: (1) RT-RPA amplification includes: a stem-loop probe that specifically recognizes microRNA, 10×RT Mix, HiScriptⅡ enzyme mixture, ERA basic, RPA upstream primer with T7 promoter, and RPA downstream primer; (2) Scaffold crRNA transcription amplification includes: split T7-1 (comprising the scaffold crRNA sequence and the complementary sequence of the T7 promoter and a complementary sequence capable of binding to half of the target RNA region), split T7-2 (comprising a partial sequence of the T7 promoter (4 nt) and a sequence capable of binding to the other half of the target RNA region), split T7-3 (comprising a partial sequence of the T7 promoter (16 nt)), T7 RNA polymerase and NTPs; (3) CRISPR system: Cas12a protein and reporter probe FQ.

2. The biosensor according to claim 1, characterized in that miR-155 and the stem-loop probe were connected and reverse transcribed into cDNA. Double-stranded T7 promoter was amplified by RPA. Then, the target RNA was transcribed under the action of T7 RNA polymerase. The target RNA was connected with split T7-1, split T7-2, and split T7-3 to form a three-way connector and a double-stranded T7 promoter with a gap site.

3. The biosensor according to claim 2, characterized in that The three-way connector begins to transcribe scaffoldcrRNA, which then binds to the sticky end of the DNA transcription template on split T7-1 to form a DNA-RNA hybrid complex. The DNA-RNA hybrid complex and scaffold crRNA are assembled together to form a function similar to split crRNA, activating Cas12a protein to cut FQ reporter and generate fluorescent signals. The complex formed by DNA-RNA acts as an activator and spacer crRNA in this system.

4. The biosensor according to claims 1-3, characterized in that: The transcribed 21nt-long RNA sequence acts as both scaffold crRNA and spacer crRNA.

5. The biosensor according to claims 1-4, characterized in that: Used for one-pot ultrasensitive and efficient detection of miR-155.

6. The use according to claim 5, characterized in that: Used for diagnosis of cancer cells and serum samples.

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