Method for detecting short-chain RNA based on crRNA random splicing mode of CRISPR-Cas12a system
By adopting the "random splicing" mode of crRNA in the CRISPR-Cas12a system, the problem of difficulty in detecting ultra-short-chain RNA in the prior art is solved, and accurate identification and sensitive detection of RNAs with lengths of 6-8 nt is achieved.
Patent Information
- Application Number
- CN202411870768.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-18
- Publication Date
- 2025-04-18
AI Technical Summary
Existing RNA detection methods are difficult to achieve sensitive detection of ultra-short-strand RNA, especially those shorter than 10 nt. Traditional methods are not ideal during reverse transcription, and the Cas13a system requires at least 28 nt of RNA to be effectively activated.
The "random splicing" crRNA based on the CRISPR-Cas12a system is adopted to cleave the crRNA from any position in the spacer to form tcrRNA and pre-hybridize with DNA activator and short-strand RNA target. The combined "random splicing" crRNA can activate Cas12a, thereby realizing the detection of short-strand RNA.
This method can effectively detect ultra-short-chain RNA with a length of 6-8 nt, achieving accurate recognition and sensitive detection of short-chain RNA, without special conditions, and has broad applicability and flexibility.
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Figure CN119842871A_ABST
Abstract
Description
Technical Field
[0002] The present invention belongs to the technical field of short-chain RNA detection, and specifically relates to a method for detecting short-chain RNA based on the crRNA "random splicing" mode of the CRISPR-Cas12a system. Background Art
[0003] Short-chain RNA plays an important role in gene expression, cell growth and development, and metabolism. The abnormal expression of some short-chain RNAs, such as microRNAs (miRNAs) and ultrashort RNAs (<10 nt), is closely related to a variety of diseases and physiological states. Therefore, the detection of short-chain RNA is of great significance for biological research and disease diagnosis.
[0004] Traditional RNA quantification methods mainly rely on reverse transcription of RNA targets into cDNA, followed by detection using various DNA amplification techniques, such as reverse transcription-polymerase chain reaction (RT-PCR), reverse transcription-loop-mediated isothermal amplification (RT-RCA), and reverse transcription-recombinase polymerase amplification (RT-RPA). However, short-chain RNA is usually a poor substrate for reverse transcription, and it is difficult to obtain high-quality cDNA. Especially those RNAs shorter than 10 nt, they cannot even be reverse transcribed. Therefore, accurate quantitative detection of ultra-short-chain RNA is a great challenge.
[0005] The CRISPR / Cas system, especially Cas12a and Cas13a, is regarded as the next generation of DNA and RNA analysis tools due to its unique targeted activated trans-cleavage activity. However, for the Cas12a sensing system, trans-cleavage is usually DNA-activated, which means that the RNA target needs to be transcribed into a DNA molecule in advance. But this reverse transcription process is not ideal for short-chain RNA, especially ultra-short-chain RNA targets. Although the Cas13a system can directly recognize and activate RNA, it requires at least 28nt of RNA to effectively activate its trans-cleavage nuclease activity, and insufficient RNA length will result in a significant decrease in detection performance. Therefore, although systems based on Cas12a and Cas13a have potential in the field of RNA detection, they have not yet solved the key challenges of sensitive detection of ultra-short-chain RNA. Summary of the invention
[0006] The purpose of the present invention is to provide a method for detecting short-chain RNA based on the crRNA "random splicing" mode of the CRISPR-Cas12a system, so as to solve the technical problem that the existing detection methods are difficult to achieve sensitive detection of ultra-short-chain RNA.
[0007] In order to achieve the above object, the present invention adopts the following technical solutions: The present invention discloses a method for detecting short-chain RNA based on the crRNA "random splicing" mode of the CRISPR-Cas12a system, comprising the following steps: The tcrRNA generated by crRNA truncation is subjected to a pre-hybridization reaction with a DNA activator and a short-chain RNA target to obtain a pre-hybridization product; The obtained pre-hybridization product, reaction buffer, Cas12a protein and fluorescent quantitative reporter chain are mixed and subjected to a cleavage reaction to obtain a reaction system solution; the fluorescent signal of the solution is then measured using a detection device.
[0008] Furthermore, tcrRNA is produced by truncating the crRNA spacer region at 2 to 15 nt.
[0009] Furthermore, tcrRNA is produced by truncating the crRNA spacer region at 4 to 12 nt.
[0010] Furthermore, the overall length of the spliced crRNA spacer obtained during the pre-hybridization reaction of tcrRNA with the DNA activator and the short RNA target is 18-40 nt; The length of the short RNA target is 6-25 nt.
[0011] Furthermore, the overall length of the spliced crRNA spacer obtained during the pre-hybridization reaction of tcrRNA with the DNA activator and the short RNA target was 18-25 nt; The length of the short RNA target is less than 10 nt.
[0012] Furthermore, the overall length of the spliced crRNA spacer obtained during the pre-hybridization reaction of tcrRNA with the DNA activator and the short RNA target was 18-25 nt; The length of the short RNA target is 6-10 nt.
[0013] Further, the concentrations of the DNA activator, tcrRNA and Cas12a protein are all 1-500 nM; The reaction buffer is 1× reaction buffer.
[0014] Furthermore, the molar ratio of Cas12a protein to tcrRNA is (0.5-2):1; the molar ratio of tcrRNA to DNA activator is 1:1; The concentration of the fluorescent quantitative reporter chain in the reaction system solution is 500 nM~2 μM.
[0015] Furthermore, the cleavage reaction is carried out at a temperature of 25-42° C. and for a time of 30-150 min.
[0016] Furthermore, the detection equipment is a fluorescence spectrometer.
[0017] Compared with the prior art, the present invention has the following beneficial effects: The present invention discloses a method for detecting short-chain RNA based on the crRNA "random splicing" mode of the CRISPR-Cas12a system. A complete Cas12a crRNA is broken from any position of the spacer region in CRISPR-Cas12a to form a truncated tcrRNA, which is then spliced with a DNA activator and a short segment of short-chain RNA. The combined "random splicing" crRNA can effectively activate Cas12a, thereby realizing the detection of short-chain RNA. The crRNA "random splicing" mode provided by the present invention allows flexible assembly of RNA targets and corresponding DNA activators, and users can fully control the length and sequence content of the splicing mode to adapt to the analysis requirements of various RNA sequences. In theory, as long as the auxiliary DNA activator sequence follows the established design principles and ensures that the lengths of the tcrRNA complementary region and the RNA targeting region are reasonable, the method can detect almost any RNA sequence. The method provides a very general and flexible solution for the accurate quantification of short-chain RNA.
[0018] Furthermore, according to relevant experimental results, the crRNA splicing method disclosed in the present invention shows almost the same Cas12a trans-cutting activation efficiency under normal conditions compared with the traditional non-splicing mode Cas12a sensing system, and does not require any special conditions. This method enables the traditional DNA-activated Cas12a system to directly and sensitively detect short-chain RNA, even ultra-short-chain RNA with a length of only 6-8 nt. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 Schematic diagram of the detection of short-chain RNA based on the crRNA "random splicing" mode of the CRISPR-Cas12a system; Among them: a-Schematic diagram of the "random splicing" mode of crRNA; b-Schematic diagram of the principle of detecting short-chain RNA targets; Figure 2 It is a comparison diagram of the relative trans-cleavage activity of Cas12a induced by the crRNA "random splicing" mode of the present invention and the conventional complete full-length crRNA; Figure 3 This is a fluorescence response diagram of detecting miRNA let-7a in the crRNA "random splicing" mode of the present invention; Figure 4 This is the fluorescence response diagram of the crRNA "random splicing" mode of the present invention detecting 8 nt p8 6s-1; Figure 5 It is a performance comparison chart of the crRNA "random splicing" mode of the present invention and the traditional Cas13a and Cas12a systems for detecting p8 6s-1s. DETAILED DESCRIPTION
[0020] In order to enable those skilled in the art to understand the characteristics and effects of the present invention, the following is a general description and definition of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used in the text are the common meanings understood by those skilled in the art for the present invention. In the event of a conflict, the definition in this specification shall prevail.
[0021] The theories or mechanisms described and disclosed herein, whether correct or incorrect, should not limit the scope of the present invention in any way, that is, the present invention can be implemented without being limited by any specific theory or mechanism.
[0022] In this article, all features such as values, quantities, contents and concentrations defined in the form of numerical ranges or percentage ranges are for simplicity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered to have included and specifically disclosed all possible secondary ranges and individual values within the range (including integers and fractions).
[0023] In this document, unless otherwise specified, “includes,” “including,” “contains,” “has,” or similar terms cover the meanings of “consisting of” and “mainly consisting of,” for example, “A includes a” covers the meanings of “A includes a and other” and “A only includes a.”
[0024] In this document, in order to make the description concise, not all possible combinations of various technical features in various embodiments or examples are described. Therefore, as long as there is no contradiction in the combination of these technical features, the various technical features in various embodiments or examples can be combined arbitrarily, and all possible combinations should be considered to be within the scope of this specification.
[0025] The present invention provides a method for detecting short-chain RNA based on the crRNA "random splicing" mode of the CRISPR-Cas12a system, in which a complete Cas12a crRNA can be broken from almost any position in the spacer region to form a truncated tcrRNA; tcrRNA alone cannot activate the trans-cleavage activity of Cas12a after binding to a DNA activator; however, when it is spliced with a short segment of short-chain RNA, the combined "random splicing" crRNA can effectively activate the trans-cleavage activity of Cas12a, thereby realizing the detection of short-chain RNA; the above process is as follows Figure 1As shown, the crRNA "random splicing" mode allows Cas12a to be activated in a wide range of splicing modes; Figure 1 As shown in a, the length of the truncated tcrRNA spacer (dark yellow) is between 3 and 15 nt; the overall length of the spliced crRNA spacer (dark yellow + pink) is between 18 and 40 nt; Figure 1 As shown in b, after tcrRNA binds to short-chain RNA targets of different lengths, it can effectively reconstruct the spacer region of crRNA, thereby forming a fully functional crRNA and activating the trans-cutting activity of Cas12a.
[0026] The method comprises the following steps: 1) In the crRNA "random splicing" assembly step, tcrRNA, DNA activator, and short-chain RNA target are pre-hybridized in the Cas12a reaction buffer at 90 °C for 5 min, and then slowly cooled to room temperature to obtain a pre-hybridization product; 2) In the Cas12a cleavage reaction step, the above pre-hybridization product, 1× reaction buffer, LbaCas12a and FQ reporter strand (5'-FAM-CCCCCCCCCC-BHQ1-3' (as shown in SEQ ID NO.1)) were subjected to cleavage reaction at 37°C for 50 min, and then heated at 90°C for 3 min to inactivate Cas12a; 3) Add 90 μL of 1× TE buffer to the above 10 μL reaction system, and measure the fluorescence signal using a F-7000 fluorescence spectrometer (Hitachi, Japan) (excitation wavelength 488 nm, emission wavelength 500-600 nm).
[0027] Preferably, tcrRNA is produced by truncating from 2 to 15 nt of the crRNA spacer region; wherein 4 to 12 nt is the optimal length of the tcrRNA spacer region.
[0028] Preferably, the overall length of the spliced crRNA spacer region is 18 to 40 nt.
[0029] Preferably, the specific concentrations of tcrRNA, DNA activator, and LbaCas12a are 1-500 nM; among which 10-50 nM is the optimal concentration.
[0030] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.
[0031] The following examples use conventional instruments and equipment in the art. The experimental methods in the following examples where specific conditions are not specified are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The various raw materials used in the following examples are conventional commercial products unless otherwise specified, and their specifications are conventional specifications in the art. In the specification of the present invention and the following examples, unless otherwise specified, "%" means weight percentage, "part" means weight part, and ratio means weight ratio.
[0032] In order to verify the versatility and flexibility of the crRNA "random splicing" mode based on the CRISPR-Cas12a system disclosed in the present invention for detecting short-chain RNA, the following experimental demonstration was performed: Example 1 Compare the relative activity of Cas12a induced by crRNA "random splicing" mode and conventional full-length crRNA: For the crRNA "random splicing" mode, equal amounts of tcrRNA-1, 12+9 DNA activator, and a 9-nt let-7a fragment (final concentration of 20 nM) were annealed at 95°C for 5 min in 1× NEBuffer 2.1 (10 mM Tris-HCl, 50 mM NaCl, 10 mMMgCl2, 100 µg / mL recombinant protein, pH 7.9), and then slowly cooled to 25°C; the same annealing procedure was used to hybridize the full-length crRNA with the 12+9 DNA activator at a molar concentration ratio of 1:1 to obtain a sample of the conventional system; In the Cas12a cleavage reaction step, the above pre-hybridization product, 1× NEBuffer 2.1, 20 nM LbaCas12a, and 1 μM FQ reporter strand (5′-FAM-CCCCCCCCCC-BHQ1-3′ (as shown in SEQ ID NO. 1)) were reacted at 37°C for 50 min, and then heated at 90°C for 3 min to inactivate Cas12a; 90 μL of 1× TE buffer (20 mM Tris-HCl, 1 mM EDTA, pH 7.9) was added to the above 10 μL reaction system, and the fluorescence signal was measured using an F-7000 fluorescence spectrometer (excitation wavelength 488 nm, emission wavelength 500-600 nm; The designed sequence is as follows: let-7a fragment: 5'-UGAGGUAGU-3' (as shown in SEQ ID NO.2); tcrRNA-1: 5'-UAAUUUCUACUAAGUGUAGAUCUGUGGGCGCGA-3' (as shown in SEQ ID NO. 3); Full-length crRNA: 5'-UAAUUUCUACUAAGUGUAGAUCUGUGGGCGCGAUGAGGUAGU-3' (as shown in SEQ ID NO. 4); 12+9 DNA activator: 5'-ACTACCTCATCGCGCCCACAG-3' (as shown in SEQ ID NO.5); The experimental results are as follows Figure 2 As shown, it can be seen that after introducing the same 12+9 DNA activator, the fluorescence intensity obtained by the "random splicing" crRNA mode of the present invention is almost the same as that produced by using the traditional full-length intact crRNA, and the induced relative trans-cutting activity is as high as nearly 100%; this result shows that by utilizing the powerful crRNA "random splicing" mechanism, the ultrashort RNA sequence can fully activate the trans-cutting function of Cas12a, and its effect is as efficient as the activity induced by the traditional complete crRNA / DNA activator structure.
[0033] Example 2 The fluorescence response of different concentrations of miRNA let-7a detected by crRNA "random splicing" mode: In the crRNA “random splicing” assembly step, 25 nM tcrRNA-1, 25 nM 12+9 DNA activator, and a series of different concentrations of miRNA let-7a were pre-hybridized in Cas12a reaction buffer (10 mM Tris-HCl, 50 mM NaCl, 12 mMMgCl2, 100 µg / mL recombinant protein, pH 7.9), reacted at 90°C for 5 min, and then slowly cooled to room temperature; In the Cas12a cleavage reaction step, the above pre-hybridization product, 1× NEBuffer 2.1, 25 nM LbaCas12a, and 1 μM FQ reporter strand (5′-FAM-CCCCCCCCCC-BHQ1-3′ (as shown in SEQ ID NO. 1)) were reacted at 37°C for 50 min, and then heated at 90°C for 3 min to inactivate Cas12a; Add 90 μL of 1× TE buffer to the above 10 μL reaction system, and measure the fluorescence signal using an F-7000 fluorescence spectrometer (excitation wavelength 488 nm, emission wavelength 500-600 nm); The designed sequence is as follows: miRNA let-7a: 5′-UGAGGUAGUAGGUUGUAUAGUU-3′ (as shown in SEQ ID NO. 6); tcrRNA-1: 5'-UAAUUUCUACUAAGUGUAGAUCUGUGGGCGCGA-3' (as shown in SEQ ID NO. 3); 12+9 DNA activator: 5'-ACTACCTCATCGCGCCCACAG-3' (as shown in SEQ ID NO.5); The experimental results are as follows Figure 3 As shown, it can be seen that as the concentration of let-7a increases from 0, 10 pM, 50 pM, 100 pM, 200 pM, 500 pM, 800 pM to 1 nM, the fluorescence response of the system gradually increases, achieving sensitive detection of miRNA let-7a.
[0034] Example 3 The fluorescence response of crRNA "random splicing" mode to detect different concentrations of 8 nt p8 6s-1: In the crRNA “random splicing” assembly step, 25 nM tcrRNA-1, 25 nM 12+9 DNA activator, and a series of different concentrations of 8 nt p8 6s-1 were pre-hybridized in the Cas12a reaction buffer, reacted at 90°C for 5 min, and then slowly cooled to room temperature; In the Cas12a cleavage reaction step, the above pre-hybridization product, 1× NEBuffer 2.1, 25 nM LbaCas12a, and 1 μM FQ reporter strand (5′-FAM-CCCCCCCCCC-BHQ1-3′ (as shown in SEQ ID NO. 1)) were reacted at 37°C for 50 min, and then heated at 90°C for 3 min to inactivate Cas12a; Add 90 μL of 1× TE buffer to the above 10 μL reaction system, and measure the fluorescence signal using an F-7000 fluorescence spectrometer (excitation wavelength 488 nm, emission wavelength 500-600 nm); The designed sequence is as follows: p8 6s-1: 5'-GUUCGGUC-3' (as shown in SEQ ID NO. 7); tcrRNA-1: 5'-UAAUUUCUACUAAGUGUAGAUCUGUGGGCGCGA-3' (as shown in SEQ ID NO. 3); 12+9 DNA activator: 5'-ACTACCTCATCGCGCCCACAG-3' (as shown in SEQ ID NO.5); The experimental results are as follows Figure 4 As shown, it can be seen that by adopting the "random splicing" mode of crRNA of the present invention, p8 6s-1 as low as 10 pM can be clearly detected; as the concentration of p8 6s-1 increases from 10 pM, 50 pM, 100 pM, 200 pM, 500 pM, 800 pM to 1 nM, the fluorescence response of the system gradually increases, achieving sensitive detection of p8 6s-1 of only 8 nt.
[0035] Example 4 Comparison of the performance of crRNA "random splicing" mode with traditional Cas13a and Cas12a systems in detecting p8 6s-1s: For the traditional Cas12a-based RNA direct detection method, the 10 μL reaction system includes 1× NEBuffer2.1, 25 nM Cas12a crRNA-p8, 25 nM LbaCas12a, 1 μM FQ reporter chain (5'-FAM-CCCCCCCCCC-BHQ1-3' (as shown in SEQ ID NO.1)), 1 nM p8 6s-1; the reaction is carried out at 37°C for 50 min, and then heated at 90°C for 3 min to inactivate Cas12a; after the reaction, 90 μL of 1× TE buffer is added to the system, and finally the fluorescence signal is measured using an F-7000 fluorescence spectrometer; For the traditional Cas13a-based RNA direct detection method, the 10 μL reaction system includes 1× reaction buffer (10 mM Tris-HCI, 1 mM DTT, 40 mM KCl, 3.5 mM MgCl2, pH=8.6), 25 nM Cas13acrRNA-p8, 25 nM LwaCas13a, 1 μM FQ reporter chain (5'-FAM-UUAUU-BHQ1-3'), 1 nM p8 6s-1; the reaction is carried out at 37°C for 50 min, and then heated at 90°C for 3 min to inactivate Cas12a; after the reaction, 90 μL of 1× TE buffer is added to the system, and finally the fluorescence signal is measured using an F-7000 fluorescence spectrometer; For the crRNA "random splicing" mode, the 10 μL reaction system includes the following two steps: in the crRNA "random splicing" assembly step, 25 nM tcrRNA-1, 25 nM 12+9 DNA activator, and 1nM p8 6s-1 are pre-hybridized in the reaction buffer of Cas12a, reacted at 90°C for 5 min, and then slowly cooled to room temperature; in the Cas12a cleavage reaction step, the above pre-hybridization product, 1× NEBuffer 2.1, 25 nM LbaCas12a and 1 μM FQ reporter chain (5'-FAM-CCCCCCCCCC-BHQ1-3' (as shown in SEQ ID NO.1)) are reacted at 37°C for 50 min, and then heated at 90°C for 3 min to inactivate Cas12a; after the reaction, 90 μL of 1× TE buffer is added to the system, and finally the F-7000 fluorescence spectrometer is used to measure the fluorescence signal; The designed sequence is as follows: p8 6s-1: 5'-GUUCGGUC-3' (as shown in SEQ ID NO. 7); Cas12a crRNA-p8: 5'-UAAUUUCUACUAAGUGUAGAUGACCGAAC-3' (as shown in SEQ ID NO. 8); Cas13a crRNA-p8: 5'-GGGAUUUAGACUACCCCAAAAACG AAGGGGACUAAAACGACCGAAC-3' (as shown in SEQ ID NO.9); tcrRNA-1: 5'-UAAUUUCUACUAAGUGUAGAUCUGUGGGCGCGA-3' (as shown in SEQ ID NO. 3); 12+9 DNA activator: 5'-ACTACCTCATCGCGCCCACAG-3' (as shown in SEQ ID NO.5); The experimental results are as follows Figure 5 As shown, the fluorescence response generated by detecting 1 nM p8 6s-1 through the crRNA "random splicing" mode is much stronger than the direct detection through the conventional Cas12a and Cas13a system; this result shows that the crRNA "random splicing" mode has obvious advantages in accurately quantifying ultrashort RNA, which is not achievable by traditional Cas12a and Cas13a systems.
[0036] The detection method disclosed in the present invention utilizes the efficient trans-cleavage activity of the CRISPR-Cas12a system, and realizes the accurate recognition of ultra-short-chain RNA through the pre-hybridization reaction of tcrRNA with DNA activator and short-chain RNA target; since the Cas12a protein will generate a strong trans-cleavage signal after recognizing the target RNA, the method has high sensitivity. At the same time, through the carefully designed tcrRNA truncation position and the length of the crRNA spacer after splicing, as well as a specific DNA activator, the method can ensure the specific recognition of the target short-chain RNA and avoid false detection; in addition, the method can detect ultra-short-chain RNA with a length of 6-8 nt, which is a challenge for traditional detection methods; Because short-chain RNA is often difficult to be stably captured and detected due to its short sequence, this method can flexibly adapt to the detection needs of ultra-short-chain RNA of different lengths and sequences by adjusting the truncation position and splicing strategy of tcrRNA; furthermore, this method has clear steps and is easy to operate, without the need for complex pretreatment steps or expensive instruments and equipment; pre-hybridization reaction, cleavage reaction and inactivation treatment can all be carried out under conventional laboratory conditions, and this method achieves real-time monitoring and quantitative analysis of the cleavage reaction by adding a fluorescent quantitative reporter chain; the intensity of the fluorescent signal is proportional to the concentration of the target short-chain RNA, so it can accurately reflect the content of the target RNA in the sample; this method has broad application prospects in biomedical research, disease diagnosis, gene therapy and other fields. Especially in terms of bacterial detection and cancer marker screening, this method can achieve sensitive, specific and rapid detection of ultra-short-chain RNA, providing strong technical support for related research and applications.
[0037] The above contents are only for explaining the technical idea of the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.
Claims
1. A method for detecting short-chain RNA based on the crRNA "random splicing" mode of the CRISPR-Cas12a system, characterized in that: The following steps are involved: The tcrRNA generated by crRNA truncation is subjected to a pre-hybridization reaction with a DNA activator and a short-chain RNA target to obtain a pre-hybridization product; The obtained pre-hybridization product, reaction buffer, Cas12a protein and fluorescent quantitative reporter chain are mixed and subjected to a cleavage reaction to obtain a reaction system solution; the fluorescent signal of the solution is then measured using a detection device.
2. A method for detecting short-chain RNA using a crRNA "random splicing" mode based on a CRISPR-Cas12a system according to claim 1, characterized in that Truncation from 2 to 15 nt of the crRNA spacer produces tcrRNA.
3. A method for detecting short-chain RNA using a crRNA "random splicing" mode based on a CRISPR-Cas12a system according to claim 2, characterized in that, Truncation from 4 to 12 nt of the crRNA spacer produces tcrRNA.
4. A method for detecting short-chain RNA using a crRNA "random splicing" mode based on a CRISPR-Cas12a system according to claim 1, characterized in that The overall length of the spliced crRNA spacer obtained during the pre-hybridization reaction of tcrRNA with DNA activators and short RNA targets is 18-40 nt; The length of the short RNA target is 6-25 nt.
5. A method for detecting short-chain RNA using a crRNA "random splicing" mode based on a CRISPR-Cas12a system according to claim 4, characterized in that, The overall length of the spliced crRNA spacer obtained during the pre-hybridization reaction of tcrRNA with DNA activators and short RNA targets is 18-25 nt; The length of the short RNA target is less than 10 nt.
6. A method for detecting short-chain RNA using a crRNA "random splicing" mode based on a CRISPR-Cas12a system according to claim 4, characterized in that The overall length of the spliced crRNA spacer obtained during the pre-hybridization reaction of tcrRNA with DNA activators and short RNA targets is 18-25 nt; The length of the short RNA target is 6-10 nt.
7. A method for detecting short-chain RNA using a crRNA "random splicing" mode based on a CRISPR-Cas12a system according to claim 1, characterized in that The concentrations of the DNA activator, tcrRNA and Cas12a protein are all 1-500 nM; The reaction buffer is 1× reaction buffer.
8. A method for detecting short-chain RNA based on the crRNA "random splicing" mode of the CRISPR-Cas12a system according to claim 1, characterized in that, The molar ratio of Cas12a protein to tcrRNA is (0.5-2):1; the molar ratio of tcrRNA to DNA activator is 1:1; The concentration of the fluorescent quantitative reporter chain in the reaction system solution is 500 nM~2 μM.
9. A method for detecting short-chain RNA using a crRNA "random splicing" mode based on a CRISPR-Cas12a system according to claim 1, characterized in that The temperature of the cleavage reaction is 25-42° C., and the time is 30-150 min.
10. A method for detecting short-chain RNA based on the crRNA "random splicing" mode of the CRISPR-Cas12a system according to claim 1, characterized in that, The detection equipment is a fluorescence spectrometer.