Method for synergistically activating CRISPR / Cas12a system based on T-type structure and application thereof

Abstract

The invention relates to the technical field of biosensing, in particular to a method for synergistically activating a CRISPR / Cas12a system based on a T-type structure and application of the method, the CRISPR / Cas12a system can be activated by utilizing two split DNA activation chains and crRNA to form the T-type structure, the activated CRISPR / Cas12a system has trans-cleavage activity, an FQ-ssDNA probe modified with an FAM fluorophore and a BHQ1 quenching group can be subjected to trans-cleavage, a fluorescence signal is recovered, and the fluorescence signal is recovered. Through reasonable design of sources of activation chains corresponding to different targets, detection targets can be expanded from traditional nucleic acid detection to non-nucleic acid targets such as proteins and metal ions, so that biosensing application of multiple targets is realized, in addition, design based on a T-type CRISPR / Cas12a synergistic activation strategy has great flexibility and universality, and the application prospect is wide. The method can be used together with various amplification strategies and detection platforms, and the sensitivity of the analysis method is effectively improved.

Description

Technical Field

[0001] The present invention relates to the field of biosensor technology, and in particular to a method for synergistically activating a CRISPR / Cas12a system based on a T-shaped structure and an application thereof. Background Art

[0002] Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are encoded by CRISPR RNA (crRNA) and are an acquired immune mechanism present in most prokaryotes. crRNA can form a complex with CRISPR associated protein (Cas), guiding the Cas protein to release endonuclease activity, thereby participating in resisting the invasion of exogenous genetic factors in bacteriophages or plasmids. In recent years, the CRISPR / Cas system has not only been used for transcriptional regulation and genome editing, but has also been widely used as a biosensor tool, playing an important role in the field of medical testing. Among them, the CRISPR / Cas12a system is considered to be one of the most influential CRISPR / Cas systems because of its excellent programmability and compatibility, and the ability to release efficient trans-cleavage activity after activation.

[0003] However, most current studies rely on the use of double-stranded DNA (dsDNA) containing a specific TTTN sequence (PAM sequence) or single-stranded DNA (ssDNA) complementary to the crRNA sequence to activate the CRISPR / Cas12a system, which reduces the programmability and flexibility of the system to a certain extent, limiting its practical application in sensing detection. The split single-stranded activation substrates that have been developed have demonstrated the possibility of synergistic activation of the CRISPR / Cas12a system. However, short split single-stranded activation substrates have poor stability and are prone to nonspecific dissociation with crRNA, which is not conducive to sensitive detection of targets.

[0004] In view of this, it is necessary to develop a stable and efficient method for co-activating the CRISPR / Cas12a system to meet the flexible application requirements of the CRISPR / Cas12a system in different fields. Summary of the invention

[0005] The object of the present invention is to provide a method and application of a CRISPR / Cas12a system based on a T-shaped structure to solve the problems of low stability and insufficient flexibility of the CRISPR / Cas12a activation system in the prior art.

[0006] To achieve the above object, the present invention provides a method for synergistically activating the CRISPR / Cas12a system based on a T-shaped structure, comprising the following steps:

[0007] Designing activation chains A and B that are partially complementary to crRNA, respectively, wherein the activation chains A and B are also partially complementary to each other, and the activation chains A, B and crRNA can be assembled into a T-shaped structure;

[0008] The designed oligonucleotide sequences of the crRNA, the activation chain A and the activation chain B are synthesized and diluted with enzyme-free water, and then vortexed and allowed to stand to obtain the diluted activation chain A, the activation chain B and the crRNA;

[0009] The diluted activation chain A, the activation chain B and the crRNA are mixed with the Cas12a protein and the ribonuclease inhibitor in a reaction buffer to obtain a reaction system, and the enzyme reaction is terminated after incubation to complete the synergistic activation of the CRISPR / Cas12a system.

[0010] Wherein, the nucleotide sequence of the activation chain A is any one of SEQ ID NO.3 to SEQ ID NO.11, and SEQ ID NO.21, SEQ ID NO.23, SEQ ID NO.25, SEQ ID NO.27, SEQ ID NO.29, SEQ ID NO.31, SEQ ID NO.33 and SEQ ID NO.35;

[0011] The nucleotide sequence of the activation chain B is any one of SEQ ID NO.12 to SEQ ID NO.20, and SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.30, SEQ ID NO.32, SEQ ID NO.34 and SEQ ID NO.36.

[0012] Among them, the nucleotide sequence of the activation chain A is SEQ ID NO.5, and the nucleotide sequence of the activation chain B is SEQ ID NO.20; the complementary part of the activation chain A and the activation chain B is 16 bases.

[0013] Wherein, the nucleotide sequence of the crRNA is SEQ ID NO.1.

[0014] Wherein, the reaction buffer is NEBuffer4.0.

[0015] The present invention also discloses a biosensor constructed based on the method as described above, the biosensor comprising Cas12a, crRNA, a starting material capable of producing activation chain A and / or activation chain B, and a reporter probe,

[0016] Wherein, the reporter probe is a FQ-ssDNA probe, and the gene sequence of the reporter probe is FAM-TTATT-BHQ1.

[0017] The present invention also discloses a method for detecting a target object using the above-mentioned biosensor, comprising the following steps:

[0018] Design and prepare the starting materials for activation chain A and activation chain B;

[0019] constructing biosensors;

[0020] The biosensor is added to the object to be tested, and the fluorescence signal is used to detect whether the object to be tested contains the target substance.

[0021] Wherein, the starting materials include hairpin H1, dsDNA, short chain d1-alkyne and short chain azide-d2.

[0022] Wherein, the target includes nucleic acid, protein and metal ion.

[0023] Wherein, the targets include miR-155, SA and divalent copper ions.

[0024] The present invention discloses a method for synergistically activating a CRISPR / Cas12a system based on a T-shaped structure and an application thereof. Based on the problem of insufficient flexibility in activating a CRISPR / Cas12a system in the prior art, a T-shaped structure is designed as an activation substrate for the CRISPR / Cas12a system. The T-shaped structure is efficient and stable, and non-specific dissociation is not likely to occur, thereby significantly improving the flexibility of the CRISPR / Cas12a system. A biosensor is also constructed to realize the application in the detection of non-nucleic acid targets such as proteins and metal ions, so as to expand the detection targets. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 It is a schematic diagram of the method for synergistically activating the CRISPR / Cas12a system based on the T-type structure of the present invention.

[0027] Figure 2 It is the fluorescence result of the different split DNA activation chain combinations of the present invention synergistically activating the CRISPR / Cas12a system.

[0028] Figure 3 It is the optimization result of the complementary base number of the split DNA activation strand, different reaction buffers and different reaction times of the present invention. The error bar represents the standard deviation of 3 repeated experiments.

[0029] Figure 4 (A) is a schematic diagram of the biosensor based on the T-type synergistic activation structure for miR-155 detection. (B) is the fluorescence result of the system with or without miR-155. (C) is the fluorescence result of the sensing system containing different concentrations of miR-155 (5pM, 10pM, 50pM, 100pM, 500pM, 1nM, 1.5nM) (the concentration increases gradually from bottom to top). (D) is the linear relationship between fluorescence intensity and miR-155 concentration between 5pM and 1.5nM. Error bars represent the standard deviation of 3 repeated experiments.

[0030] Figure 5 (A) is a schematic diagram of the scheme of the biosensor based on the T-type synergistic activation structure for SA detection. (B) is the fluorescence result of the system with or without SA. (C) is the fluorescence result of the sensing system containing different concentrations of SA (1nM, 2.5nM, 5nM, 7.5nM, 10nM, 12.5nM, 15nM) (the concentration of the curve increases from bottom to top). (D) is the linear relationship between fluorescence intensity and SA concentration between 1nM and 15nM. The error bar represents the standard deviation of 3 repeated experiments.

[0031] Figure 6 (A) shows a biosensor based on a T-type cooperative activation structure for Cu 2+ Schematic diagram of the detection scheme. (B) is the presence or absence of Cu in the system 2+ (C) is the fluorescence results of the samples containing different concentrations of Cu 2+ (10pM, 50pM, 100pM, 500pM, 750pM, 1nM, 1.5nM) of the sensing system (the concentration increases from bottom to top). (D) is the fluorescence intensity and Cu 2+ The linear relationship was observed between 10 pM and 1.5 nM. Error bars represent the standard deviation of three replicate experiments. DETAILED DESCRIPTION

[0032] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limiting the present invention.

[0033] See also Figures 1 to 3 , the present invention provides a method for synergistically activating the CRISPR / Cas12a system based on a T-type structure, and the activation ability of the T-type structure can be finely adjusted by the position and length of two DNA activation substrate chains. When the T-type structure does not exist, the CRISPR / Cas12a system cannot be activated. When the two split DNA activation chains are successfully assembled into a T-type structure with crRNA, the CRISPR / Cas12a system can be activated, resulting in non-specific trans-cleavage of the FQ-ssDNA probe modified with a FAM fluorescent group and a BHQ1 quenching group, and the fluorescence signal is restored.

[0034] The scheme of the present invention is described below in conjunction with the accompanying drawings, wherein the experimental methods without specific operating steps are all carried out in accordance with the corresponding product instructions, and the instruments, reagents, and consumables used in the examples can all be purchased from commercial companies unless otherwise specified.

[0035] Example 1: Method for synergistically activating the CRISPR / Cas12a system based on T-shaped structure and application of biosensor.

[0036] 1. Materials:

[0037] EnGen LbaCas12a (Cpf1), Klenow fragment (3'-5'exo-, KF), Nicking enzyme (Nt.BsmAI), 10×NEBufferr2.1, 10×rCutsmart, 10×NEBuffer2.0, 10×NEBuffer3.0 and 10×NEBuffer4.0 were purchased from New England Biolabs (Ipswich, MA, USA). CuSO4·5H2O was purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). BTTAA was purchased from MedChemExpress (Beijing, China). HPLC-purified crRNA, recombinant RNase inhibitor, dNTPs, streptavidin, ascorbic acid, HPLC-purified oligonucleotide chains, and enzyme-free water were purchased from Sangon Biotechnology Co., Ltd (Shanghai, China).

[0038] 2. Instruments

[0039] All fluorescence spectra were recorded using a fluorescence spectrophotometer F-4700 (HATACHI, Japan) with an excitation wavelength of 490 nm and an emission wavelength of 520 nm; the slits at the inlet and outlet were both 5 nm. The DNA suspension was quantified using a NanoDrop1000 spectrophotometer (Thermo Scientific, USA). The reaction system was incubated in a mini metal bath (HANU MINIC-100).

[0040] 3. Method Construction

[0041] like Figure 1 As shown, the composition based on the T-type CRISPR / Cas12a synergistic activation method includes Cas12a, crRNA, activation chain A, activation chain B and FQ-ssDNA probe, wherein the activation chain A, activation chain B and crRNA are assembled to form a T-type structure, successfully activating the CRISPR / Cas12a system.

[0042] In the process of T-type structure synergistic activation of CRISPR / Cas12a system, first, enzyme-free water was added to the oligonucleotide sequence synthesized in Table 1, vortexed for 3min, and then kept still for 3min at 4°C, and repeated three times. The concentrations of activation chain A (100μM) and activation chain B (100μM) were diluted to 1μM; the concentration of crRNA (100μM) was diluted to 1μM; the concentration of FQ-ssDNA probe (100μM) was diluted to 50μM. Subsequently, the diluted activation chain A, activation chain B, crRNA, Cas12a protein, FQ-ssDNA probe and ribonuclease inhibitor were mixed in the reaction buffer to obtain a 100μL reaction system, and the enzyme reaction was terminated by incubating at 37°C for 60min and then at 65°C for 10min (the final concentrations of activation chain A and activation chain B in the system were 10nM, the final concentration of crRNA was 50nM, and the concentration of FQ-ssDNA probe was 500nM). Finally, the fluorescence emission intensity at 520 nm was measured using a fluorescence spectrometer, the excitation wavelength was 490 nm, the scanning wavelength range was set to 500-600 nm, and both the excitation slit and the emission slit were set to 5 nm.

[0043] The results are as follows Figure 2 As shown in the figure, by comparing the fluorescence signals of different split DNA activator combinations that synergistically activate the CRISPR / Cas12a system, it can be found that when the activation chain A8 and the activation chain B14 form a T-shaped structure, the activation efficiency of the CRISPR / Cas12a system is the best. Subsequently, the optimal combination of the activation chain A8 and the activation chain B14 was optimized for the number of complementary bases, reaction buffer, and reaction time. The results are shown in the figure. Figure 3As shown, when the stem of the T-type activation structure, i.e., the part (C part) where the activation chain A8 and B14 are hybridized, is optimized for the number of bases, it is found that the signal-to-noise ratio is optimal when A8 and B14 complement 16 bases. Then, the T-type structure pre-assembly time is optimized, and it can be seen that the assembly time is 0, i.e., the signal-to-noise ratio is optimal when the one-step reaction is directly added. Then, different reaction buffers are replaced to conduct experiments, and it is determined that the signal-to-noise ratio of the one-step method in the NEBuffer4.0 system is optimal. Finally, the reaction time of the one-step method is optimized under the optimal conditions, and it can be seen that the one-step method 60min can obtain the highest activation efficiency. The above experimental results demonstrate the successful construction of the method for synergistically activating the CRISPR / Cas12a system based on the T-type structure, and the method can be used for subsequent application development. It is known that when the T-type structure does not exist, the CRISPR / Cas12a system cannot be activated, and the T-type structure is successfully formed by rationally designing the source of the activation chain, and the activation of the CRISPR / Cas12a system and the cracking of the FQ-ssDNA probe can be achieved, and the effective recovery of the fluorescence signal can be detected by a fluorescence spectrometer.

[0044] By rationally designing the source of activation chains corresponding to different targets, this method can be used to detect different target molecules. Therefore, the design based on the T-type CRISPR / Cas12a synergistic activation strategy has great flexibility. The design of this sensing strategy can expand the detection targets from traditional nucleic acid detection to non-nucleic acid targets such as proteins and metal ions, and due to the versatility of the design, it can be used in conjunction with a variety of amplification strategies and detection platforms to effectively improve the sensitivity of the analytical method.

[0045] Table 1 DNA and RNA sequences involved in the examples

[0046]

[0047]

[0048] See also Figures 4 to 6 The present invention also discloses a method for detecting a target object using a biosensor.

[0049] Embodiment 2:

[0050] Application of T-type synergistic activation structure-based biosensor in miR-155 detection.

[0051] By rationally designing the hairpin H1 from the activation strand B, a biosensor based on a T-type cooperative activation structure was constructed for the detection of miR-155. Figure 4As shown in A, during a typical strand displacement amplification (SDA) cycle reaction, miR-155 hybridizes with the template hairpin H1 to form a structure that can be recognized by the KF polymerase. The KF enzyme can polymerize to the 3' end of the template H1 to form a completely complementary dsDNA, thereby providing a recognition site for the Nt.BsmAI enzyme (a sequence-specific shearing enzyme). The Nt.BsmAI enzyme can generate a cleavage site on the extension domain of miR-155, and then the KF enzyme recognizes the cleavage site to initiate secondary extension. During the secondary extension process, the KF enzyme has strand displacement activity and can release the previously generated ssDNA (activation chain B) into the solution. Therefore, under the joint action of the KF enzyme and the Nt.BsmAI enzyme, abundant activation chain B can be obtained to participate in the subsequent CRISPR / Cas12a system reaction.

[0052] In the detection of miR-155, hairpin H1 was first prepared by annealing, and the specific steps were as follows: hairpin H1 was kept at 95°C in a thermal cycler for 5-10 min, cooled to 4°C within 5 h, and taken out and stored at 4°C for use. Different concentrations of miR-155, hairpin H1 (500nM), activation chain A (1μM), crRNA (1μM), Cas12a protein (1μM), KF enzyme (5U / μL), Nt.BsmAI enzyme (5U / μL), dNTPs (10mM), FQ-ssDNA probe (50μM) and ribonuclease inhibitor were mixed in the reaction buffer, incubated at 37°C for 30min and then incubated at 65°C for 10min to terminate the enzyme reaction, and the detection system was completed with a total of 100μL (the final concentration of hairpin H1 was 50nM, the final concentration of activation chain A was 25nM, the final concentration of crRNA was 50nM, and the concentration of FQ-ssDNA probe was 500nM). The control group did not add miR-155 but added an equal volume of enzyme-free water, and the other conditions were the same as the experimental group. The oligonucleotide sequences used in the above experiments are shown in Table 2. Finally, the fluorescence emission intensity at 520 nm was measured using a fluorescence spectrometer, the excitation wavelength was 490 nm, the scanning wavelength range was set to 500-600 nm, and both the excitation slit and the emission slit were set to 5 nm.

[0053] The results are as follows Figure 4 As shown in Figure B, by comparing the fluorescence signals of the sensing system with and without the target miR-155, it can be found that in the absence of miR-155, the sensing system is in a closed state, producing a relatively low background fluorescence. On the contrary, in the presence of miR-155, significant fluorescence recovery can be observed, proving the feasibility of the proposed method for miR-155 detection.

[0054] like Figure 4As shown in CD, under the optimal experimental conditions (reaction temperature of 37 degrees and reaction time of 30 minutes), the fluorescence intensity of the sensing system increased with the increase of the target miR-155 concentration, and within the miR-155 concentration range of 5pM to 1.5nM (R 2 =0.9926) showed a good linear relationship, and the regression equation was Y=31.67+2.11X. The detection limit (LOD) was calculated to be 5.41pM. The entire test could be completed within 30min, achieving rapid and sensitive detection.

[0055] Table 2 DNA and RNA sequences involved in the examples

[0056]

[0057] Embodiment 3:

[0058] Application of biosensor based on T-type cooperative activation structure in SA detection.

[0059] By rationally designing two groups of dsDNA (BKA and BKB) derived from activation chain A and activation chain B, a biosensor based on a T-type cooperative activation structure was constructed for the detection of SA. Figure 5 As shown in A, in the absence of SA, activation strands A and B are blocked by two sets of dsDNA (BKA and BKB). When SA is present, the activation strands A and B can be displaced by the proximity ligation effect (PLA) to participate in the formation of the subsequent T-type synergistic activation structure, synergistically activate the CRISPR / Cas12a system, and achieve a fluorescent signal response.

[0060] In the detection of SA, the closed dsDNA (BKA and BKB) was first prepared by annealing. The specific steps were: keep the activation chain A and the closed chain bkA, the activation chain B and the closed chain bkB in a thermal cycler at 95°C for 5-10min, cool to 4°C within 5h, and take out and store at 4°C for use. SA, BKA (1μM), BKB (1μM), crRNA (1μM), Cas12a protein (1μM), FQ-ssDNA probe (50μM) and ribonuclease inhibitors of different concentrations were mixed in the reaction buffer, incubated at 37°C for 60min, and then incubated at 65°C for 10min to terminate the enzyme reaction, and the detection system was completed. A total of 100μL (the final concentration of BKA and BKB was 10nM, the final concentration of crRNA was 50nM, and the concentration of FQ-ssDNA probe was 500nM) was constructed. The control group did not add SA but added an equal volume of enzyme-free water, and the other conditions were the same as the experimental group. The oligonucleotide sequences used in the above experiments are shown in Table 3. Finally, the fluorescence emission intensity at 520 nm was measured using a fluorescence spectrometer, the excitation wavelength was 490 nm, and the scanning wavelength range was set to 500-600 nm. Both the excitation slit and the emission slit were set to 5 nm.

[0061] The results are as follows Figure 5 As shown in Figure B, by comparing the fluorescence signals of the sensing system with and without the target SA, it can be found that in the absence of SA, the background fluorescence signal is low and the sensing system is in a closed state. On the contrary, in the presence of SA, the fluorescence signal is significantly restored, proving the feasibility of the proposed method for SA detection.

[0062] like Figure 5 CD shows that under the optimal experimental conditions (reaction temperature of 37 degrees and reaction time of 60 minutes), the fluorescence intensity of the sensing system increases with the increase of the target SA concentration, and in the SA concentration range of 1nM to 15nM (R 2 =0.9956) showed a good linear relationship, and the regression equation was Y=-88.37+114.05X. The detection limit (LOD) was calculated to be 62.35 pM. The entire reaction was completed within 60 min, achieving sensitive detection.

[0063] Table 3 DNA and RNA sequences involved in the examples

[0064]

[0065]

[0066] Embodiment 4:

[0067] Biosensor based on T-type cooperative activation structure in Cu 2+ Application in detection.

[0068] By rationally designing the short-chain d1-alkyne and azide-d2 derived from the activation chain B and utilizing the specific copper click chemistry reaction, a biosensor based on a T-type synergistic activation structure was constructed for the detection of Cu 2+ .like Figure 6 As shown in A, ascorbic acid can reduce Cu(II) to Cu(I), and Cu(I) can catalyze the azide-alkyne cycloaddition (CuAAC) click chemistry reaction to connect the two short chains d1-alkyne and azide-d2 to form a d1-d2 complex. In the final signal output process, with the help of crRNA and activation chain A, the d1-d2 complex can participate in the formation of a T-type synergistic activation structure, activate the CRISPR / Cas12a system, and achieve a fluorescent signal response. Through this sensing strategy, Cu 2+ The concentration was positively correlated with the fluorescence intensity.

[0069] In Cu 2+ In the detection, different concentrations of Cu 2+ , 1μLd1-alkyne (5μM), 1μLazide-d2 (5μM), 5μL ascorbic acid (10mM), 2.4μL BTTAA (5mM) were mixed in the reaction buffer and reacted at room temperature for 30min. Subsequently, activation chain A (1μM), crRNA (1μM), Cas12a protein (1μM), FQ-ssDNA probe (50μM) and ribonuclease inhibitor were added to the above reaction system, incubated at 37°C for 60min, and then incubated at 65°C for 10min to terminate the enzyme reaction, completing the construction of a total of 100μL of the detection system (the final concentration of activation chain A was 10nM, the final concentration of crRNA was 50nM, and the concentration of FQ-ssDNA probe was 500nM). The control group did not add Cu 2+ However, an equal volume of enzyme-free water was added, and other conditions were the same as the experimental group. The oligonucleotide sequences used in the above experiments are shown in Table 4. Finally, the fluorescence emission intensity at 520 nm was measured using a fluorescence spectrometer, the excitation wavelength was 490 nm, and the scanning wavelength range was set to 500-600 nm. The excitation slit and emission slit were both set to 5 nm.

[0070] The results are as follows Figure 6 As shown in B, by comparing the presence and absence of the target Cu 2+ The fluorescence signal of the sensing system can be found in the absence of Cu 2+ In the case of Cu 2+ In the presence of Cu, the fluorescence signal was significantly restored, demonstrating the applicability of the proposed method to Cu 2+ Feasibility of detection.

[0071] like Figure 6 CD shows that under the optimal experimental conditions (reaction temperature of 37 degrees and reaction time of 60 minutes), the fluorescence intensity of the sensing system increases with the target Cu 2+ The concentration of Cu increased and the 2+ Concentration range (R 2 =0.9931) showed a good linear relationship, and the regression equation was Y=-2.50+0.69X. The detection limit (LOD) was calculated to be 14.42pM. The entire reaction was completed within 60min, achieving sensitive detection.

[0072] Table 4 DNA and RNA sequences involved in the examples

[0073]

[0074] The method of the present invention, which is based on the T-shaped structure to synergistically activate the CRISPR / Cas12a system, breaks away from the traditional limitation of the CRISPR / Cas12a system on the recognition of dsDNA containing PAM sequences, expands the versatility and scope of its application, and only requires the rational design of the activation chain source of the T-shaped structure to achieve rapid detection of multiple targets, showing great advantages in sensitivity and specificity.

[0075] What is disclosed above is only a preferred embodiment of the present invention, and it certainly cannot be used to limit the scope of rights of the present invention. Ordinary technicians in this field can understand that all or part of the processes of the above embodiment and equivalent changes made according to the claims of the present invention still fall within the scope of the invention.

Claims

1. A method for synergistically activating a CRISPR / Cas12a system based on a T-type structure, characterized in that: The steps include: Designing activation chains A and B that are partially complementary to crRNA, respectively, wherein the activation chains A and B are also partially complementary to each other, and the activation chains A, B and crRNA are assembled into a T-shaped structure; The designed oligonucleotide sequences of the crRNA, the activation chain A and the activation chain B are synthesized and diluted with enzyme-free water, and then vortexed and allowed to stand to obtain the diluted activation chain A, the activation chain B and the crRNA; The diluted activation chain A, the activation chain B and the crRNA are mixed with the Cas12a protein and the ribonuclease inhibitor in a reaction buffer to obtain a reaction system, and the enzyme reaction is terminated after incubation to complete the synergistic activation of the CRISPR / Cas12a system.

2. The method for synergistically activating the CRISPR / Cas12a system based on a T-shaped structure as claimed in claim 1, characterized in that The nucleotide sequence of the activation chain A is any one of SEQ ID NO.3 to SEQ ID NO.11, and SEQ ID NO.21, SEQ ID NO.23, SEQ ID NO.25, SEQ ID NO.27, SEQ ID NO.29, SEQ ID NO.31, SEQ ID NO.33 and SEQ ID NO.35; The nucleotide sequence of the activation chain B is any one of SEQ ID NO.12 to SEQ ID NO.20, and SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.30, SEQ ID NO.32, SEQ ID NO.34 and SEQ ID NO.

36.

3. The method for synergistically activating the CRISPR / Cas12a system based on the T-shaped structure as claimed in claim 2, characterized in that The nucleotide sequence of the activation chain A is SEQ ID NO.5, and the nucleotide sequence of the activation chain B is SEQ ID NO.20; The complementary part between the activation chain A and the activation chain B is 16 bases.

4. The method for synergistically activating the CRISPR / Cas12a system based on the T-shaped structure as claimed in claim 1, characterized in that The nucleotide sequence of the crRNA is SEQ ID NO.

1.

5. The method for synergistically activating the CRISPR / Cas12a system based on the T-shaped structure as claimed in claim 1, characterized in that The reaction buffer is NEBuffer 4.

0.

6. A biosensor constructed based on the method according to any one of claims 1 to 5, characterized in that: The biosensor includes Cas12a, crRNA, a starting material that can produce activation chain A and / or activation chain B, and a reporter probe. Wherein, the reporter probe is a FQ-ssDNA probe, and the gene sequence of the reporter probe is FAM-TTATT-BHQ1.

7. A method for detecting a target object using the biosensor according to claim 6, characterized in that: The steps include: Design and prepare the starting materials of activation chain A and activation chain B through the target; constructing biosensors; Adding the biosensor to the object to be tested; The fluorescence signal is used to determine whether the object to be detected contains the target substance.

8. The detection method according to claim 7, characterized in that The starting materials include hairpin H1, dsDNA, short chain d1-alkyne and short chain azide-d2.

9. The detection method according to claim 7, characterized in that: The targets include nucleic acids, proteins and metal ions.

10. The detection method according to claim 9, characterized in that: The targets include miR-155, SA and divalent copper ions.

Images