Method for activating CRISPR (clustered regularly interspaced short palindromic repeats)-Cas enzyme by hot start and application of method in nucleic acid detection

Abstract

The invention relates to a method for activating CRISPR-Cas enzyme through hot start and application of the method in nucleic acid detection, and belongs to the technical field of gene detection. The invention provides a method for controlling the enzyme activity of Cas in a CRISPR-Cas system through temperature, which comprises the following steps: adding single-stranded DNA (deoxyribonucleic acid) into the CRISPR-Cas system, inhibiting the enzyme activity of the Cas under the condition of temperature 1, and activating the enzyme activity of the Cas under the condition of temperature 2; under the condition of the temperature 1, the single-chain DNA is complementarily paired with a hand and / or spacer region base in the crRNA to form closed crRNA; the closed crRNA is thermally denatured into a single chain under the condition of the temperature 2. The Cas enzyme activity can be started by controlling the temperature, and accurate time sequence regulation and control of amplification in a single tube and CRISPR reaction are achieved. Operation is simple and equipment dependence is low. The cost is low; and the design difficulty is low.

Description

Technical Field

[0001] This invention relates to the field of gene detection technology, and in particular to a method for hot-start activation of CRISPR-Cas enzyme and its application in nucleic acid detection. Background Technology

[0002] CRISPR (clustered regularly spaced short palindromic repeats) are nucleic acid sequences in bacteria and archaea associated with the adaptive immune response system. CRISPR-associated proteins (Cas) are endonucleases that form complexes with crRNA (CRISPR RNA). The crRNA recognizes the target nucleic acid, guiding the Cas protein to recognize and cleave the target sequence; this enzymatic activity is called cis-cleavage activity. Class II CRISPR-Cas systems, such as Cas12 and Cas13, form a Cas-crRNA-target DNA ternary complex that, after cis-cleavage, exhibits trans-cleavage activity, enabling non-specific cleavage of nearby single-stranded DNA or RNA. Combining this property with FRET (fluorescence resonance transfer) technology enables nucleic acid detection. An oligonucleotide sequence (reporter) with a fluorescent group and a fluorescence quencher at its ends, after cis-cleavage in a CRISPR-Cas system, generates a fluorescent signal through trans-cleavage of the reporter. Because CRISPR-Cas recognizes one target DNA or RNA, it can trans-cleave thousands of reporters, achieving signal amplification and enabling nucleic acid detection. Cas12 can specifically recognize and detect DNA, while Cas13 can specifically recognize and detect RNA.

[0003] In addition, isothermal amplification technology, with its characteristics of being fast, accurate, specific, and not dependent on sophisticated instruments, has shown good application prospects in clinical and rapid diagnosis. Common types include recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), ligation amplification (SDA), and rolling circle amplification (RCA).

[0004] The CRISPR-Cas system, with its precise ability to identify and cleave nucleic acid sequences, has enormous application potential in the field of nucleic acid detection. However, direct detection of nucleic acids using the CRISPR-Cas system suffers from low sensitivity. Combining amplification technology with the CRISPR-Cas system can achieve specific identification of amplified products, further improving the accuracy and sensitivity of detection. For example, the novel nucleic acid detection methods SHERLOCK and DETECTR, developed using the CRISPR-Cas system as the core and combined with RPA isothermal amplification technology, exhibit high sensitivity and specificity. This combined technology has become an important research direction in the field of pathogen detection.

[0005] Traditional CRISPR nucleic acid detection technology separates isothermal amplification and the CRISPR reaction into two steps, with the CRISPR-Cas enzyme added only after amplification. However, this requires opening the container, increasing the probability of cross-contamination, and is inconvenient to operate.

[0006] Currently, a single-tube method integrates isothermal amplification with CRISPR-Cas in a single reaction system. Taking RPA as an example, at an RPA amplification temperature of around 37°C, the CRISPR-Cas-crRNA complex binds to the target nucleic acid and activates Cas enzyme activity before RPA amplification. The activated Cas protein degrades the target nucleic acid through cis-cleavage, preventing RPA amplification and affecting detection sensitivity, leading to false negatives. Similarly, combining CRISPR with other isothermal amplification technologies presents the same problem: the two reaction processes interfere with each other, making it difficult to guarantee detection stability and repeatability, thus affecting detection efficiency.

[0007] Currently, two new technologies can address the temporal regulation of isothermal amplification and CRISPR reactions: one is techniques that physically separate the amplification and CRISPR reactions; the other is techniques that combine CRISPR and Cas systems. The technique of physically separating the amplification and CRISPR reactions utilizes a "tube-in-tube" structure to spatially separate the two systems. Once the concentration of the amplified product is increased, it is transferred as a whole to the CRISPR system to initiate the reaction. The technique of combining CRISPR and Cas systems achieves temporal regulation by pre-inhibiting Cas enzyme activity, encompassing approaches including functional blocking of crRNA, direct inhibition of the Cas enzyme, and photoregulation.

[0008] Physical separation technology relies on a specific "tube-in-tube" physical structure, which increases the complexity and cost of the experimental setup and requires high precision in device fabrication, hindering its widespread application in primary healthcare institutions and on-site testing. Furthermore, the transfer efficiency of amplified products is easily affected by various factors, resulting in imprecise timing of the CRISPR reaction initiation.

[0009] Chemical and molecular regulation techniques also have their own problems. For example, chemical modification regulation requires chemical modification of crRNA, increasing the difficulty and cost of synthesis. The removal efficiency of photostable groups is affected by light conditions, which may lead to incomplete recovery or overactivation of crRNA activity, affecting detection accuracy. Strict storage conditions are required, limiting its application in on-site detection. Photodegradation primer technology requires additional light equipment and operating steps, increasing the complexity of the experimental process and dependence on equipment. The photodegradation process is affected by factors such as light intensity and time, easily leading to unstable primer degradation efficiency, which in turn affects the synergy of RPA and CRISPR reactions and reduces detection repeatability. Regulation of Cas protein binding balance via heparin sodium is easily affected by other components in the reaction system, especially unknown and complex components in clinical samples, which may lead to insufficient or excessive inhibition, reducing detection reliability. Summary of the Invention

[0010] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for hot-start activation of CRISPR-Cas enzyme and its application in nucleic acid detection.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a method for controlling the activity of Cas enzyme in a CRISPR-Cas system by temperature, characterized in that single-stranded DNA is added to the CRISPR-Cas system to inhibit Cas enzyme activity at temperature 1 and to activate Cas enzyme activity at temperature 2. Under the specified temperature 1 condition, the single-stranded DNA and the handle and / or spacer regions of the crRNA undergo complementary base pairing to form a closed crRNA. The crRNA blocked at temperature 2 is thermally denatured into a single strand.

[0012] Existing amplification techniques coupled with CRISPR-Cas systems for pathogen detection suffer from problems such as poor reaction timing control, complex operation, high cost, poor stability and reproducibility, and limited applicability. This invention addresses these issues by designing single-stranded DNA that is complementary to the handle and / or spacer regions of crRNA. At low temperatures, the crRNA is blocked with this single-stranded DNA, forming blocked crRNA that inhibits Cas enzyme activity in the CRISPR-Cas system. At high temperatures, the blocked crRNA is denatured into single strands, activating Cas enzyme activity. This process controls the CRISPR reaction, ensuring the accuracy of nucleic acid detection.

[0013] Furthermore, some bases in the single-stranded DNA are modified with locked nucleic acids and / or methoxy groups.

[0014] Furthermore, the Cas enzyme is selected from any one of Cas12a, Cas12b, and Cas13a enzymes.

[0015] Furthermore, temperature 1 is lower than temperature 2, where temperature 2 is the activation temperature of the Cas enzyme. The temperature can be adjusted according to the reaction requirements by changing the length of the nucleic acid sequence in the blocked region of the blocked crRNA and the degree of modification.

[0016] In a preferred embodiment of the present invention, the temperature 2 is greater than 40°C or 60°C, and RPA amplification or LAMP amplification is performed under the temperature 1 condition.

[0017] In a more preferred embodiment of the present invention, the temperature 1 is 35~38°C for RPA amplification, and the temperature 2 is 40~42°C for CRISPR-Cas12a reaction.

[0018] In a more preferred embodiment of the present invention, temperature 1 is 35°C for RPA amplification and temperature 2 is 42°C for CRISPR-Cas12a reaction.

[0019] In a more preferred embodiment of the present invention, temperature 1 is 50~55°C for LAMP amplification, and temperature 2 is 60~65°C for CRISPR-Cas12b reaction.

[0020] In a more preferred embodiment of the present invention, temperature 1 is 55°C for LAMP amplification, and temperature 2 is 60°C for CRISPR-Cas12b reaction.

[0021] Secondly, the present invention provides a one-pot hot-start nucleic acid detection method combining RPA amplification and CRISPR-Cas12a-mediated activation, comprising the following steps: S11: Mix single-stranded DNA and crRNA, anneal at 90-95℃ to obtain blocked crRNA; in the blocked crRNA, the single-stranded DNA and the handle and / or spacer regions of the crRNA are complementary base pairs. S12: Mix Cas12a enzyme, RPA reaction reagent, Cas12a-reporter, target gene, and blocked crRNA to obtain the RPA-CRISPR-Cas12a reaction system. First, incubate at 35-38℃ to maintain the blocked state of the crRNA and perform RPA pre-amplification. Then, heat to 40-45℃ to release the blocked state of the crRNA, activate Cas12a enzyme activity, perform CRISPR-Cas12a reaction, and detect the fluorescence intensity of the target gene.

[0022] Furthermore, the gene to be tested contains a specific fragment with a nucleotide sequence as shown in SEQ ID NO: 1 or 13; the nucleotide sequence of the crRNA is shown in SEQ ID NO: 11; and the nucleotide sequence of the single-stranded DNA is shown in any one of SEQ ID NO: 2 to 10.

[0023] In a preferred embodiment of the present invention, some bases in the single-stranded DNA are modified with locked nucleic acids and / or methoxy groups.

[0024] In a more preferred embodiment of the present invention, in the single-stranded DNA with the nucleotide sequence shown in SEQ ID NO: 2, the 3rd base at the 5' end is modified with LNA (locked nucleic acid), and the 4th base is modified with 2'-OMe (2'-methoxy)RNA. This is a single-stranded DNA complementary to the 9nt bases in the spacer region of crRNA.

[0025] In a more preferred embodiment of the present invention, the nucleotide sequence of the single-stranded DNA as shown in SEQ ID NO: 3 is such that the 7th and 11th bases at the 5' end are modified with 2'-OMe (2'-methoxy)RNA. This is a single-stranded DNA complementary to the 12-nt spacer region of the crRNA.

[0026] In a more preferred embodiment of the present invention, the single-stranded DNA with the nucleotide sequence shown in SEQ ID NO: 4 has the 5' end bases at positions 3, 5, 7, 9, 11, 13, 15, and 17 modified with 2'-OMe (2'-methoxy)RNA. This is a single-stranded DNA complementary to the 18-nt spacer region of the crRNA.

[0027] In a more preferred embodiment of the present invention, the single-stranded DNA with the nucleotide sequence shown in SEQ ID NO: 5 is a single-stranded DNA with 3 nt bases complementary to the intact handle region and the spacer region of crRNA. The single-stranded DNA with the nucleotide sequence shown in SEQ ID NO: 6 is a single-stranded DNA with 5 nt bases complementary to the intact handle region and the spacer region of crRNA. The single-stranded DNA with the nucleotide sequence shown in SEQ ID NO: 7 is a single-stranded DNA with 8 nt bases complementary to the intact handle region and the spacer region of crRNA. The single-stranded DNA with the nucleotide sequence shown in SEQ ID NO: 8 is a single-stranded DNA with 11 nt bases complementary to the intact handle region and the spacer region of crRNA. The single-stranded DNA with the nucleotide sequence shown in SEQ ID NO: 9 is a single-stranded DNA with 14 nt bases complementary to the intact handle region and the spacer region of crRNA.

[0028] In a more preferred embodiment of the present invention, in the single-stranded DNA with the nucleotide sequence shown in SEQ ID NO: 10, the 3rd and 12th bases at the 5' end are modified with LNA (locked nucleoside), and the 5th, 10th, 13th, and 14th bases are modified with 2'-OMe (2'-methoxy)RNA. This is a single-stranded DNA complementary to the intact handle region of crRNA.

[0029] Further, in step S11, 0.7 μl of 1.25 μM single-stranded DNA and 0.6 μl of 1.25 μM crRNA are mixed and incubated at 90–95 °C for 1–5 min, then slowly cooled to room temperature to obtain blocked crRNA. Incubation at 90 °C for 3 min is preferred.

[0030] In a preferred embodiment of the present invention, in step S12, the incubation is first carried out at 35°C, and then the temperature is raised to 42°C for further incubation.

[0031] In a preferred embodiment of the present invention, in step S12, the temperature is first raised to 35°C for 5-15 minutes, preferably 15 minutes; then the temperature is raised to 42°C for 10-60 minutes, preferably 60 minutes.

[0032] Furthermore, in step S12, the RPA-CRISPR-Cas12a reaction system also contains BSA and Mg(CH3COO)2.

[0033] Further, in step S12, the nucleotide sequence of the Cas12a-reporter is as shown in SEQ ID NO: 12, and the 5' end of the Cas12a-reporter contains a HEX group and the 3' end contains a BHQ1 group.

[0034] Further, in step S12, the RPA reaction reagent is a conventional commercially available RPA reaction reagent, containing a forward primer, a reverse primer, Buffer V, RAA lyophilized particles, and water; the nucleotide sequence of the forward primer is shown in SEQ ID NO: 14, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO: 15.

[0035] Further, in step S12, the RPA reaction reagent contains 2 μl of 10 μM forward primer, 2 μl of 10 μM reverse primer, 25 μl of Buffer V, and 15 μl of purified water.

[0036] Further, in step S12, 7.2 μl of RPA reaction reagent, 0.5 μl of 3 μM LbCas12a, 0.6 μl of 12.5 μM Cas12a-reporter, and 0.5 μl of 7.5 mg / ml BSA are mixed, and blocked crRNA and 0.5 μl of 250 aM of the target gene are added. Then, 1.225 μl of Mg(CH3COO)2 is added to activate the reaction, mixed well, and placed in a PCR instrument for fluorescence detection.

[0037] Thirdly, the present invention provides a one-pot hot-start nucleic acid detection method combining LAMP amplification and CRISPR-Cas12b-mediated activation, comprising the following steps: S21: Mix single-stranded DNA and crRNA, anneal at 90-95℃ to obtain blocked crRNA; in the blocked crRNA, the single-stranded DNA and the handle region of the crRNA are complementary base pairs. S22: Mix Cas12b enzyme, LAMP reaction reagent, Cas12b-reporter, target gene, and blocked crRNA to obtain the LAMP-CRISPR-Cas12b reaction system. First, incubate at 50-55℃ to maintain the blocked state of the crRNA and perform LAMP pre-amplification. Then, raise the temperature to 60-65℃ to release the blocked state of the crRNA, activate Cas12b enzyme activity, perform CRISPR-Cas12b reaction, and detect the fluorescence intensity of the target gene.

[0038] Furthermore, the gene to be tested contains a specific fragment with a nucleotide sequence as shown in SEQ ID NO: 16; the nucleotide sequence of the crRNA is shown in SEQ ID NO: 18; and the nucleotide sequence of the single-stranded DNA is shown in SEQ ID NO: 17.

[0039] Further, in step S21, 0.7 μl of 1.25 μM single-stranded DNA and 0.6 μl of 1.25 μM crRNA are mixed and incubated at 90–95 °C for 1–5 min, then slowly cooled to room temperature to obtain blocked crRNA. Incubation at 90 °C for 3 min is preferred.

[0040] In a preferred embodiment of the present invention, in step S22, the temperature is first incubated at 55°C, and then raised to 60°C for further incubation.

[0041] In a preferred embodiment of the present invention, in step S22, the temperature is first raised to 55°C for 5-15 minutes, preferably 15 minutes; then the temperature is raised to 60°C for 10-60 minutes, preferably 60 minutes.

[0042] Furthermore, in step S22, the LAMP-CRISPR-Cas12b reaction system also contains RNase inhibitors and MgCl2.

[0043] Further, in step S22, the nucleotide sequence of the Cas12b-reporter is as shown in SEQ ID NO: 12, and the 5' end of the Cas12b-reporter contains a HEX group and the 3' end contains a BHQ1 group.

[0044] Further, in step S22, the LAMP reaction reagent is a conventional commercially available LAMP reaction reagent containing WarmStartLAMP 2X Master Mix, FIP_primer, BIP_primer, F3_primer, B3_primer, LF_primer, and BF_primer. The nucleotide sequence of FIP_primer is shown in SEQ ID NO: 19, the nucleotide sequence of BIP_primer is shown in SEQ ID NO: 20, the nucleotide sequence of F3_primer is shown in SEQ ID NO: 21, the nucleotide sequence of B3_primer is shown in SEQ ID NO: 22, the nucleotide sequence of LF_primer is shown in SEQ ID NO: 23, and the nucleotide sequence of BF_primer is shown in SEQ ID NO: 24.

[0045] Furthermore, in step S22, the LAMP reaction reagent contains 1.25 μl of primer premix and 6.25 μl of WarmStart LAMP 2X Master Mix.

[0046] Furthermore, in step S22, 5 μl of 16 μM FIP_primer, 5 μl of 16 μM BIP_primer, 5 μl of 2 μM LF_primer, 5 μl of 2 μM LB_primer, 5 μl of 4 μM F3_primer, and 5 μl of 4 μM B3_primer are mixed to prepare a primer premix.

[0047] Furthermore, in step S22, 7.5 μl of LAMP reaction reagent is mixed with 0.5 μl of 1.25 μM Cas12b, 0.6 μl of 12.5 μM Cas12b-reporter, 0.35 μl of 40 U / μl RNase inhibitor, and 1.25 μl of 100 mM MgCl2. Blocked crRNA is added, and 1 μl of 25 aM of the gene to be tested is added. Fluorescence detection is then performed in a PCR instrument.

[0048] Fourthly, the present invention provides a one-pot hot-start nucleic acid detection kit combining RPA amplification and CRISPR-Cas12a-mediated activation, characterized in that the kit contains Cas12a enzyme, RPA reaction reagent, Cas12a-reporter, crRNA and single-stranded DNA, wherein the single-stranded DNA is complementary to the handle and / or spacer regions of the crRNA.

[0049] Furthermore, the nucleotide sequence of the crRNA is shown in SEQ ID NO: 11, and the nucleotide sequence of the single-stranded DNA is shown in any one of SEQ ID NO: 2 to 10, and some bases in the single-stranded DNA are modified with locked nucleic acids and / or methoxy groups.

[0050] Furthermore, the nucleotide sequence of the Cas12a-reporter is shown in SEQ ID NO: 12, and the Cas12a-reporter contains a HEX group at its 5' end and a BHQ1 group at its 3' end.

[0051] Furthermore, the kit also contains BSA and Mg(CH3COO)2.

[0052] Furthermore, the RPA reaction reagent contains a forward primer, a reverse primer, Buffer V, RAA lyophilized particles, and water; the nucleotide sequence of the forward primer is shown in SEQ ID NO: 14, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO: 15.

[0053] Fifthly, the present invention provides a one-pot hot-start nucleic acid detection kit combining LAMP amplification and CRISPR-Cas12b-mediated activation, characterized in that the kit contains Cas12b enzyme, LAMP reaction reagent, Cas12b-reporter, crRNA and single-stranded DNA, wherein the single-stranded DNA is complementary to the handle region of the crRNA.

[0054] Furthermore, the nucleotide sequence of the crRNA is shown in SEQ ID NO: 18, and the nucleotide sequence of the single-stranded DNA is shown in SEQ ID NO: 17.

[0055] Furthermore, the nucleotide sequence of the Cas12b-reporter is shown in SEQ ID NO: 12, and the Cas12b-reporter contains a HEX group at its 5' end and a BHQ1 group at its 3' end.

[0056] Furthermore, the kit also contains RNase inhibitors and MgCl2.

[0057] Furthermore, the LAMP reaction reagent contains WarmStart LAMP 2X Master Mix, FIP_primer, BIP_primer, F3_primer, B3_primer, LF_primer, and BF_primer. The nucleotide sequence of FIP_primer is shown in SEQ ID NO: 19, the nucleotide sequence of BIP_primer is shown in SEQ ID NO: 20, the nucleotide sequence of F3_primer is shown in SEQ ID NO: 21, the nucleotide sequence of B3_primer is shown in SEQ ID NO: 22, the nucleotide sequence of LF_primer is shown in SEQ ID NO: 23, and the nucleotide sequence of BF_primer is shown in SEQ ID NO: 24.

[0058] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention can activate Cas enzyme activity by controlling the temperature, thereby achieving precise timing regulation of single-tube amplification and CRISPR reaction. The crRNA can be blocked at low temperature and unblocked at high temperature, ensuring smooth amplification in a single tube first. After amplification is completed, Cas enzyme activity is activated to carry out CRISPR reaction, avoiding mutual interference between the two and ensuring the accuracy of detection.

[0059] (2) Simple operation and low equipment dependence. Hot start is convenient and quick, without the need for complex physical structures such as "tube-in-tube" or special operating steps such as additional light. Only the reaction temperature needs to be controlled. The entire detection process can be completed with a single tube, which reduces the dependence on special equipment and is more conducive to application in primary medical institutions and on-site testing.

[0060] (3) Low cost and easy design. No chemical modification of crRNA is required. The design of closed single-stranded DNA sequences reduces the cost and difficulty of synthesis, and also avoids the problem of poor storage stability of modified crRNA. The design of primers and other sequences is relatively simple, and there is no need to optimize the matching of multiple regions at the same time, which lowers the threshold for technology application. Attached Figure Description

[0061] Figure 1 This is a schematic diagram of the thermally activated CRISPR-Cas12a enzyme trans-cleavage reaction.

[0062] Figure 2 This diagram illustrates the principle of the thermally activated CRISPR-Cas12a enzyme cis-cleavage reaction, using a single-stranded DNA sequence complementary to the middle region of the crRNA-spacer as an example.

[0063] Figure 3 This diagram illustrates the principle of the thermally activated CRISPR-Cas12a enzyme cis-cleavage reaction, using a single-stranded DNA sequence complementary to the complete crRNA-handle region and the extended crRNA-spacer region as an example.

[0064] Figure 4This section presents the effects of ssDNA_spacer_middle_9nt, ssDNA_spacer_middle_12nt, and ssDNA_spacer_18nt_2'-OMe on the detection of target DNA at different temperatures. Specifically, A shows the fluorescence growth curves of 100pM target DNA when ssDNA_spacer_middle_9nt is involved in the reaction at different temperatures, where 's' represents ssDNA_spacer_middle_9nt. The color bars only display the corresponding curve colors: blue for 34℃, green for 38℃, orange for 41℃, and red for 45℃. B shows the statistical graph of Cas12a enzyme trans-cleavage activity when ssDNA_spacer_middle_9nt is involved in the reaction at different temperatures. C shows the fluorescence growth curves of 100pM target DNA when ssDNA_spacer_middle_12nt is involved in the reaction at different temperatures. The graph shows the Cas12a enzyme trans-cleavage activity at different temperatures. Here, 's' represents ssDNA_spacer_middle_12nt, and the color bars only display the corresponding curve colors: blue for 34℃, green for 38℃, orange for 41℃, and red for 45℃. 'D' shows the statistical graph of Cas12a enzyme trans-cleavage activity at different temperatures when ssDNA_spacer_middle_12nt participates in the reaction. 'E' shows the fluorescence growth curve of 100pM target DNA when ssDNA_spacer_18nt_2'-OMe participates in the reaction at different temperatures. 'F' shows the statistical graph of Cas12a enzyme trans-cleavage activity at different temperatures when ssDNA_spacer_18nt_2'-OMe participates in the reaction.

[0065] Figure 5 The cis-cleavage activity statistics are shown for ssDNA_spacer_middle_9nt, ssDNA_spacer_middle_12nt, and ssDNA_spacer_18nt_2'-OMe participating in the reaction at 38℃.

[0066] Figure 6This image shows the trans-cleavage activity of the Cas12a enzyme at different temperatures when ssDNA_handle_extend_3nt, ssDNA_handle_extend_5nt, ssDNA_handle_extend_8nt, ssDNA_handle_extend_11nt, and ssDNA_handle_extend_14nt are involved in the reaction. Specifically, A shows the fluorescence growth curve of 100pM target DNA when ssDNA_handle_extend_3nt, ssDNA_handle_extend_5nt, ssDNA_handle_extend_8nt, ssDNA_handle_extend_11nt, and ssDNA_handle_extend_14nt are involved in the reaction at 38℃; B shows the fluorescence growth curve of ssDNA_handle_extend_3nt, ssDNA_handle_extend_5nt, ssDNA_handle_extend_8nt, ssDNA_handle_extend_11nt, and ssDNA_handle_extend_14nt are involved in the reaction at 38℃. C is a statistical graph of the trans-cleavage activity of Cas12a enzyme when ssDNA_handle_extend_14nt is involved in the reaction; D is a statistical graph of the trans-cleavage activity of Cas12a enzyme when ssDNA_handle_extend_8nt is involved in the reaction at different temperatures; E is a statistical graph of the fluorescence growth of 100pM target DNA when ssDNA_handle_extend_11nt is involved in the reaction at different temperatures; F is a statistical graph of the trans-cleavage activity of Cas12a enzyme when ssDNA_handle_extend_11nt is involved in the reaction at different temperatures.

[0067] Figure 7 This is a statistical chart of cis-cleavage activity when ssDNA_handle_extend_3nt, ssDNA_handle_extend_5nt, ssDNA_handle_extend_8nt, ssDNA_handle_extend_11nt, and ssDNA_handle_extend_14nt participate in the reaction at 38℃.

[0068] Figure 8This section shows the trans-cleavage activity of the Cas12a enzyme when ssDNA_handle_2'-OMe_LNA is involved in the reaction at different temperatures. In section A, the fluorescence growth curves of 100pM target DNA are shown when ssDNA_handle_2'-OMe_LNA is involved in the reaction at different temperatures; blue represents 36℃, green 38℃, orange 40℃, and red 42℃. Section B is a statistical graph of the trans-cleavage activity of the Cas12a enzyme at different temperatures when ssDNA_handle_2'-OMe_LNA is involved in the reaction.

[0069] Figure 9 This is a nucleic acid detection method using a one-pot hot-start reaction of RPA and CRISPR with ssDNA containing blocking crRNA. The figure only shows an example of ssDNA blocking complementary to the spacer region of crRNA.

[0070] Figure 10 The results of the nucleic acid detection method using a combined RPA and CRISPR one-pot hot-start reaction with ssDNA containing blocking crRNA are compared with the fluorescence curves of the traditional one-pot reaction.

[0071] Figure 11 This is a schematic diagram of the thermally activated CRISPR-Cas12b enzyme reaction.

[0072] Figure 12 The images show the trans-cleavage activity of the Cas12b enzyme when ssDNA_handle is involved in the reaction at different temperatures. A shows the fluorescence growth curve of 100pM target DNA detected when ssDNA_handle is involved in the reaction at 55℃; B shows the comparison of fluorescence intensity at the reaction endpoint when ssDNA_handle is involved in the reaction at different temperatures.

[0073] Figure 13 This is a nucleic acid detection method that combines LAMP and CRISPR one-pot hot-start reaction with ssDNA containing blocking crRNA.

[0074] Figure 14 The results of the nucleic acid detection method using a combined LAMP and CRISPR one-pot hot-start reaction with ssDNA containing blocking crRNA are compared with the fluorescence curves of the traditional one-pot reaction.

[0075] Figure 15 This is a schematic diagram of the thermally activated CRISPR-Cas13a enzyme reaction.

[0076] Figure 16The images show the trans-cleavage activities of the Cas13a enzyme at different temperatures when ssDNA_handle, ssDNA_handle_all_extend_12nt, and ssDNA_all are involved in the reaction. Specifically, A shows the fluorescence growth curves of 10pM target RNA detected when ssDNA_handle, ssDNA_handle_all_extend_12nt, and ssDNA_all are involved in the reaction at 38℃; B shows the statistical graph of the trans-cleavage activities of the Cas13a enzyme at 38℃ when ssDNA_handle, ssDNA_handle_all_extend_12nt, and ssDNA_all are involved in the reaction; C shows the fluorescence growth curves of 10pM target RNA detected when ssDNA_all is involved in the reaction at different temperatures; and D shows the statistical graph of the trans-cleavage activities of the Cas13a enzyme when ssDNA_all is involved in the reaction at different temperatures. Detailed Implementation

[0077] To better illustrate the purpose, technical solution, and advantages of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, other materials and reagents used in the embodiments are commercially available.

[0078] Example 1: Design of ssDNA blocking crRNA to inhibit Cas12a protease activity I. Experimental Methods 1. Design of ssDNA to block crRNA This invention uses specific ssDNA to pre-block a portion of crRNA, thereby inhibiting Cas enzyme activity and preventing premature activation of Cas enzyme, which would lead to premature degradation of the target DNA or RNA and prevent amplification, thus ensuring the smooth progress of the amplification reaction. After amplification is complete, the ssDNA blocking of crRNA is released by regulating the temperature, activating Cas enzyme activity and enabling specific recognition and cleavage of the amplification product.

[0079] To achieve better blocking of crRNA by ssDNA at lower temperatures, this invention pre-designs an ssDNA sequence to block crRNA based on the melting temperature (Tm) and verifies the trans-cleavage activity of the Cas12a enzyme at different temperature gradients. The crRNA can be divided into two parts: a crRNA-handle and a crRNA-spacer. The crRNA-handle binds to the Cas enzyme, while the crRNA-spacer binds to the target sequence.

[0080] The designed ssDNA sequence, target DNA, crRNA sequence, and Cas12a-reporter sequence are shown in Table 1. Specifically, ssDNA_spacer_middle_9nt is a single-stranded DNA sequence complementary to the 9nt middle region of the crRNA-spacer; ssDNA_spacer_middle_12nt is a single-stranded DNA sequence complementary to the 12nt middle region of the crRNA-spacer; ssDNA_spacer_18nt_2'-OMe is a single-stranded DNA sequence complementary to the 18nt region of the crRNA-spacer; and ssDNA_handle_extend_3nt and ssDNA_handle_extend_3nt are also shown in Table 1. A_handle_extend_5nt, ssDNA_handle_extend_8nt, ssDNA_handle_extend_11nt, and ssDNA_handle_extend_14nt are single-stranded DNA sequences complementary to the complete crRNA-handle region and the extended portion of the crRNA-spacer region, respectively (3nt, 5nt, 8nt, 11nt, and 14nt represent the length of the extension, respectively); ssDNA_handle_2'-OMe_LNA is a single-stranded DNA sequence complementary to the complete crRNA-handle region, containing locked nucleic acid modified and 2'-methoxy modified bases.

[0081] Table 1 In Table 1, underlined nucleic acids represent those modified with LNA (locked nucleosides), while bolded nucleic acids represent those modified with 2'-OMe (2'-methoxy) RNA. The core of 2'-methoxy modification is the 2'-hydroxyl group (-OH) on the ribose ring of the nucleic acid, which replaces the hydrogen atom in the hydroxyl group with a methyl group (-CH3), forming a 2'-O-methylated structure. Therefore, the base T in the ssDNA_spacer_18nt_2'-OMe sequence is designed to be the base U.

[0082] 2. Trans-cleavage activity experiment (schematic diagram as shown) Figure 1 (As shown), the steps are as follows: (1) Blocking: Incubate 1.25 μl crRNA (600 nM) with 1.25 μl sDNA sequence (700 nM) or DEPC water (blank control, 1.25 μl) at 90 °C for 3 min, and then slowly cool to room temperature to obtain a blocked crRNA mixture.

[0083] (2) Preparation of reaction system: Mix 1.25 μl of LbCas12a (500 nM), 1.25 μl of NEBuffer r2.1 (10×), 0.75 μl of Cas12a-reporter (10 μM), 0.5 μl of BSA (7.5 mg / ml), 1.25 μl of MgCl2 (100 mM) and 4.375 μl of DEPC water containing 0.1% (v / v) Triton in a tube, add the blocked crRNA mixture and 0.625 μl of Target DNA (2 nM, i.e. the final target concentration is 100 pM), and transfer the resulting reaction system (12.5 μl) to a PCR instrument for fluorescence detection.

[0084] (3) Fluorescence detection: Set up the HEX fluorescence channel, incubate the reaction system at different temperatures for 1 h, detect the fluorescence intensity every 1 min, and process the collected signals.

[0085] Note: LbCas12a enzyme was purchased from New England Biolabs (Shanghai, China).

[0086] 3. Cis-cutting activity experiment (schematic diagram as shown) Figure 2 and Figure 3 As shown, these correspond to different ssDNA target sites. The difference from the trans-cleavage activity assay reaction system is that it does not contain Cas12a-reporter, and the volume of DEPC water containing 0.1% (v / v) Triton is 4.5 μl, the target DNA is 1.25 μl, and the concentration is 2 μM, meaning the final target concentration is 200 nM. All other parameters are the same. Fluorescence detection: The HEX fluorescence channel was set, and the reaction system was incubated at 38°C for 1 h. The fluorescence intensity was measured every 1 min, and the collected signals were processed.

[0087] II. Experimental Results 1. Figure 4 A shows the fluorescence growth curves of 100pM target DNA detected after blocking crRNA with ssDNA_spacer_middle_9nt at different temperatures. At 34℃, 38℃, 41℃, and 45℃, the fluorescence intensity of the ssDNA blocking crRNA was almost no different from the blank control group, indicating that it did not effectively block crRNA. This may be due to the short design of the blocking ssDNA sequence, leading to unstable binding. Even if it can pair complementaryly with a portion of the crRNA region, it still cannot achieve a blocking effect. The fluorescence intensity at the end of the fluorescence growth curve characterizes the trans-cleavage activity of the Cas12a enzyme, and the corresponding results are shown below. Figure 4 As shown in B.

[0088] 2. For example Figure 4 As shown in Figure C, the fluorescence growth curves of 100pM target DNA were detected after blocking crRNA with ssDNA_spacer_middle_12nt at different temperatures. At 34℃, 38℃, 41℃, and 45℃, the final fluorescence intensity of this ssDNA was slightly lower than that of the blank control group, indicating that it has a blocking effect on crRNA, but the effect is weak. The trans-cleavage activity of the Cas12a enzyme was characterized by the fluorescence intensity at the end of the fluorescence growth curve, and the corresponding results are shown in Figure C. Figure 4 As shown in D.

[0089] 3. Figure 4 E represents the fluorescence growth curves of 100pM target DNA detected after blocking crRNA with ssDNA_spacer_18nt_2'-OMe at different temperatures. The blocking effects on crRNA were similar at 36℃ and 38℃, and superior to those at 40℃ and 42℃. The blocking effect gradually decreased with increasing temperature, and the trans-cleavage activity of the Cas12a enzyme gradually recovered to the unblocked state. The blocking effect at this stage was also characterized by the fluorescence intensity at the endpoint of the fluorescence intensity curve, as shown in the figure. Figure 4 As shown in F, ssDNA_spacer_18nt_2'-OMe, as a single-stranded DNA nucleic acid targeting the crRNA spacer region, is well-suited for subsequent amplification techniques and CRISPR one-pot hot-start reactions.

[0090] Furthermore, to investigate the inhibitory mechanism of ssDNA on CRISPR-Cas12a enzyme activity, a cis-cleavage experiment was conducted. At 38℃, cis-cleavage assays were performed on three ssDNA strands: ssDNA_spacer_middle_9nt, ssDNA_spacer_middle_12nt, and ssDNA_spacer_18nt_2'-OMe. The results showed that ssDNA_spacer_18nt_2'-OMe exhibited the best inhibitory effect, consistent with the inhibitory effect observed in the trans-cleavage assay. The corresponding results are shown below. Figure 5 As shown.

[0091] 4. (1) Figure 6A represents the fluorescence growth curve of 100pM target DNA detected after blocking crRNA with ssDNA_handle_extend_3nt, ssDNA_handle_extend_5nt, ssDNA_handle_extend_8nt, ssDNA_handle_extend_11nt, and ssDNA_handle_extend_14nt at 38℃. The blocking effects of ssDNA_handle_extend_8nt and ssDNA_handle_extend_11nt were optimal. Therefore, temperature gradient experiments were performed on these two blocked ssDNA lines, and the results are as follows. Figure 6 C and Figure 6 As shown in Figure E, ssDNA_handle_extend_11nt almost completely blocked crRNA at different temperature gradients; ssDNA_handle_extend_8nt showed similar blocking effects on crRNA at 36℃ and 38℃, and was superior to 40℃ and 42℃. Its blocking effect gradually weakened with increasing temperature, and the trans-cleavage activity of Cas12a enzyme gradually recovered to the unblocked state. The blocking effect at this stage was characterized by the fluorescence intensity at the endpoint of the fluorescence intensity curve, and the corresponding results are shown in Figure E. Figure 6 B. Figure 6 D and Figure 6 As shown in F, ssDNA_handle_extend_8nt is well-suited for subsequent amplification techniques and CRISPR one-pot hot-start reactions.

[0092] (2) To investigate the inhibitory mechanism of ssDNA on CRISPR-Cas12a enzyme activity, a cis-cleavage experiment was further conducted. At 38℃, cis-cleavage was performed on five ssDNA strands: ssDNA_handle_extend_3nt, ssDNA_handle_extend_5nt, ssDNA_handle_extend_8nt, ssDNA_handle_extend_11nt, and ssDNA_handle_extend_14nt. The results showed that the inhibitory effect increased with increasing ssDNA length, a trend consistent with the inhibitory effect observed in the trans-cleavage experiment. The corresponding results are as follows: Figure 7 As shown.

[0093] 5. (1) This invention designs ssDNA to block only the crRNA-handle region, but Figure 6Results A demonstrate that simply using ordinary complementary DNA to block the crRNA-handle region is insufficient to guarantee the blocking effect. Therefore, modified nucleic acids are used to improve affinity, including methoxy-modified and locked nucleic acid-modified blocking ssDNA, and ssDNA_handle_2'-OMe_LNA is designed.

[0094] (2) Figure 8 A shows the fluorescence growth curves of 100pM target DNA detected after blocking crRNA with ssDNA_handle_2'-OMe_LNA at different temperatures. ssDNA_handle_2'-OMe_LNA is a single-stranded DNA sequence with the same theoretical Tm value as ssDNA_spacer_18nt_2'-OMe and ssDNA_handle_extend_8nt. The trans-cleavage activity of the Cas12a enzyme is characterized by the fluorescence intensity at the endpoint of the fluorescence growth curve, and the corresponding results are shown below. Figure 8 As shown in B.

[0095] Example 2: Nucleic acid detection method based on RPA and CRISPR-Cas12a one-pot hot-start reaction I. Experimental Methods Based on the ssDNA sequence of the blocked crRNA obtained in Example 1, the Cas12a enzyme can be activated at around 40°C, which matches the RPA amplification reaction temperature. Therefore, it was further applied to the CRISPR-Cas12a one-pot hot-start reaction combined with RPA. The following experiments were performed using ssDNA_spacer_18nt_2'-OMe as the ssDNA of the blocked crRNA.

[0096] The nucleic acid sequences used in the one-pot hot-start experiment based on RPA and CRISPR are shown in Table 2. Due to the long sequence of TargetdsDNA, only one single-stranded sequence from the double-stranded sequence is given in Table 2. The RPA kit used in the experiment was purchased from Biolifesci (catalog number M20901-0048), and its main components are RAA lyophilized particles (i.e., the basic reaction unit, including the key proteins required to drive RPA amplification and the basic components to maintain a stable amplification environment), purified water, Buffer V, and Mg(CH3COO)2.

[0097] Table 2 Note: The target DNA was based on the Mycobacterium tuberculosis H37Rv reference genome numbered AL123456.3 in the GenBank database. The target region studied was the segment of nucleotides from position 890105 to position 890312 on that genome.

[0098] The experimental steps for the one-pot hot-start reaction of RPA and CRISPR are as follows (the principle is as follows). Figure 9 (as shown) (1) Preparation of RPA premix: Place the RAA (recombinase-mediated isothermal nucleic acid amplification) lyophilized particles from the kit on ice and centrifuge at 1000 rpm for 30 s before use to ensure that the powder settles to the bottom.

[0099] According to the RPA components in the kit instructions, add 2 μl of Forward_primer (10 μM), 2 μl of Reverse_primer (10 μM), 25 μl of Buffer V and 15 μl of purified water to the tube containing RAA lyophilized particles, centrifuge at 1000 rpm for 10 s to confirm that all components are completely dissolved, and obtain the RPA premix.

[0100] (2) Blocking: Incubate 0.6 μl crRNA (1.25 μM) and 0.7 μl ssDNA (1.25 μM) at 90 °C for 3 min, then slowly cool to room temperature to obtain a blocked crRNA mixture.

[0101] (3) Preparation of reaction system: Mix 7.2 μl of RPA premix, 0.5 μl of LbCas12a (3 μM), 0.6 μl of Cas12a-reporter (12.5 μM) and 0.5 μl of BSA (7.5 mg / ml) in a tube, add the blocked crRNA mixture and 0.5 μl of Target DNA (250 aM, i.e. the final target concentration is 10 aM), then add 1.225 μl of Mg (CH3COO)2 to activate the reaction, gently pipette to mix (avoid air bubbles), and immediately place in a PCR instrument for fluorescence detection.

[0102] (4) Fluorescence detection: Set up the HEX fluorescence channel, incubate at 35℃ for 15 min and then at 42℃ for 75 min, detect the fluorescence intensity every 1 min, and process the collected signals.

[0103] II. Experimental Results Figure 10The fluorescence growth curves of the CRISPR-Cas12a one-pot hot-start reaction with ssDNA containing blocked crRNA and RPA are compared with those of the conventional one-pot reaction. Real-time fluorescence detection showed that the CRISPR-Cas12a one-pot hot-start reaction with ssDNA containing blocked crRNA and RPA can detect the target rapidly and accurately. In contrast, the conventional CRISPR-Cas12a one-pot reaction with RPA produced almost no signal, and its fluorescence intensity was consistent with the negative control (NTC). The difference between the conventional one-pot reaction and the CRISPR-Cas12a one-pot hot-start reaction with ssDNA containing blocked crRNA and RPA is that: (1) ssDNA is replaced with an equal volume of DEPC water; (2) the separate blocking step is omitted, and all reaction system reagents are directly mixed in the same tube, while the other operating parameters remain the same.

[0104] In summary, the CRISPR-Cas12a one-pot hot-start reaction based on ssDNA containing blocked crRNA combined with RPA exhibits higher sensitivity and better detection performance compared to traditional one-pot methods.

[0105] Example 3: Nucleic acid detection method using a CRISPR-Cas12b one-pot hot-start reaction with ssDNA containing blocked crRNA. I. Experimental Methods Following the design materials of Example 1, an ssDNA sequence complementary to the handle region of crRNA was designed. In Example 1, the handle region of crRNA was relatively short, making it difficult for the designed ssDNA to block Cas12a activity through the complementary crRNA handle region; however, the crRNA bound to Cas12b has a relatively longer handle region, allowing for the blocking of Cas12b enzyme activity through the complementary crRNA handle region.

[0106] The nucleic acid sequences used in the trans-cleavage activity assay are shown in Table 3. Due to the long sequence of the Target DNA in Table 3, only one single-stranded sequence from the double-stranded sequence is given.

[0107] Table 3 Trans-cleavage activity experiment (schematic diagram as follows) Figure 11 (As shown), the steps are as follows: (1) Blocking: First, incubate 1.25 μl of crRNA (600 nM) with 1.25 μl of ssDNA sequence (700 nM) or DEPC water (blank control, 1.25 μl) at 90 °C for 3 min, and then slowly cool to room temperature to obtain the blocked crRNA mixture.

[0108] (2) Preparation of reaction system: Mix 1.25 μl of AapCas12b (500 nM), 1.25 μl of NEBuffer r3.1 (10x), 0.75 μl of Cas12b-reporter (10 μM), 0.35 μl of RNase inhibitor (40 U / μl), 1.25 μl of MgCl2 (100 mM) and 4.525 μl of DEPC water containing 0.1% Triton in a tube, add the blocked crRNA mixture and 0.625 μl of Target DNA (2 nM, i.e. the final target concentration is 100 pM), and transfer the resulting system (12.5 μl) to a PCR instrument for fluorescence detection.

[0109] (3) Fluorescence detection: Set up the HEX fluorescence channel, incubate the reaction system at different temperatures for 1 h, detect the fluorescence intensity every 1 min, and process the collected signals.

[0110] Note: Cas12b (AapCas12b) of Bacillus acidophilus was purchased from Bio-lifesci.

[0111] II. Experimental Results Figure 12 A shows the fluorescence growth curve of Cas12b detecting 100pM target nucleic acid at 55℃ under blocking conditions containing ssDNA_handle. The ssDNA_handle effectively blocks Cas12b enzyme activity at 55℃. Therefore, experiments were conducted on this ssDNA at 55℃ and 60℃ respectively, and the endpoint fluorescence intensity of the fluorescence growth curves was compared (results are shown in Figure 1). Figure 12 As shown in B), it was found that ssDNA_handle almost completely blocked the Cas12b trans enzyme activity at 55℃; when the temperature was raised to 60℃, its blocking effect was significantly weakened, and the trans cleavage activity of the Cas12b enzyme returned to the unblocked state. Moreover, the blocking effect of this ssDNA can reach more than 90%, which meets the expected ideal blocking effect.

[0112] Example 4: Nucleic Acid Detection Method Based on LAMP and CRISPR One-Pot Hot-Start Reaction I. Experimental Methods Based on the ssDNA sequence of the blocked crRNA from Example 3, the Cas12b enzyme, which can be activated at around 60°C, matches the LAMP amplification reaction temperature. Therefore, it was further applied to a one-pot hot-start CRISPR-Cas12b reaction combined with LAMP. If the target is RNA, it needs to be reverse transcribed to obtain cDNA before LAMP amplification (i.e., RT-LAMP). The nucleic acid sequences used in the CRISPR-Cas12b one-pot hot-start reaction of ssDNA containing blocking crRNA and combined with LAMP are shown in Table 4. Due to the long length of the target DNA sequence, only one single-stranded sequence from the double-stranded sequence is given in Table 4. The WarmStart® LAMP Kit (DNA & RNA) used in the experiment was purchased from New England Biolabs. The WarmStart LAMP 2X Master Mix reagent contains the key proteins required to drive LAMP amplification and the basic components needed to maintain a stable amplification environment.

[0113] Table 4 Note: The target DNA was based on the SARS-CoV-2 reference genome numbered NC-045512 in the GenBank database. The target region studied was the segment of nucleotides from position 13201 to position 15600 on that genome.

[0114] One-pot hot-start reaction of LAMP and CRISPR (schematic diagram shown) Figure 13 (As shown), the steps are as follows: (1) Preparation of LAMP premix: First, mix 6 primers together, including 5 μl FIP_primer (16 μM), 5 μl BIP_primer (16 μM), 5 μl LF_primer (2 μM), 5 μl LB_primer (2 μM), 5 μl F3_primer (4 μM) and 5 μl B3_primer (4 μM), to obtain primer premix; then mix 1.25 μl of primer premix with 6.25 μl of WarmStart LAMP 2X Master Mix in the kit to obtain LAMP premix.

[0115] (2) Blocking: Incubate 0.6 μl of crRNA (1.25 μM) with 0.7 μl of ssDNA sequence (1.25 μM) or DEPC water (blank control, 0.7 μl) at 90 °C for 3 min, and then slowly cool to room temperature to obtain a blocked crRNA mixture.

[0116] (3) Preparation of reaction system: Mix 7.5 μl of LAMP premix with 0.5 μl of AapCas12b (1.25 μM), 0.6 μl of Cas12b-reporter (12.5 μM), 0.35 μl of RNase inhibitor (40 U / μl), and 1.25 μl of MgCl2 (100 mM) in a tube, add the blocked crRNA mixture, add 1 μl of Target DNA (25 aM, i.e. the final target concentration is 2 aM), and transfer the resulting reaction system (12.5 μl) to a PCR instrument for fluorescence detection.

[0117] (4) Fluorescence detection: Set up the HEX fluorescence channel, incubate at 55℃ for 15 min and then at 60℃ for 75 min, collect the signal once every 1 min, and process the collected signal.

[0118] II. Experimental Results Figure 14 The fluorescence growth curves of the CRISPR-Cas12b one-pot hot-start reaction with ssDNA containing blocked crRNA and LAMP are shown below. The differences between the traditional one-pot reaction and the CRISPR-Cas12b one-pot hot-start reaction with ssDNA containing blocked crRNA and LAMP are: (1) replacing ssDNA with an equal volume of DEPC water; (2) omitting the separate blocking step and directly mixing all reaction system reagents in the same tube, while keeping other operating parameters consistent. Real-time fluorescence detection showed that the CRISPR-Cas12b one-pot hot-start reaction with ssDNA containing blocked crRNA and LAMP can rapidly and accurately detect target DNA. In contrast, the traditional one-pot reaction produced almost no signal, and its fluorescence intensity was consistent with the negative control (NTC). In summary, the CRISPR-Cas12b one-pot hot-start reaction based on ssDNA containing blocked crRNA and LAMP exhibits higher sensitivity and better detection performance compared to the traditional one-pot method.

[0119] Example 5: Nucleic acid detection method using a one-pot hot-start CRISPR-Cas13a reaction with ssDNA containing blocked crRNA. I. Experimental Methods Following the design principles of Example 1, ssDNA sequences complementary to a portion of the crRNA region were designed, namely ssDNA_handle, ssDNA_handle_all_extend_12nt, and ssDNA_all.

[0120] The nucleic acid sequences used in the trans-cleavage activity assay are shown in Table 5.

[0121] Table 5 Trans-cleavage activity experiment (schematic diagram as follows) Figure 15 (As shown), the steps are as follows: (1) Blocking: First, incubate 1.25 μl of crRNA (600 nM) with 1.25 μl of ssDNA sequence (700 nM) or DEPC water (blank control, 1.25 μl) at 90 °C for 3 min, and then slowly cool to room temperature to obtain the blocked crRNA mixture.

[0122] (2) Preparation of reaction system: Mix 1.25 μl of LbuCas13a (500 nM), 1.25 μl of Cas13a Buffer (10×), 1.25 μl of Cas13a-reporter (10 μM), 0.32 μl of RNase inhibitor (40 U / μl), 0.5 μl of MgCl2 (87.5 mM) and 4.18 μl of DEPC water containing 0.1% Triton in a tube, add the blocked crRNA mixture and 1.25 μl of Target RNA (100 pM, i.e. the final target concentration is 10 pM), transfer the resulting system (12.5 μl) to the PCR instrument for fluorescence detection.

[0123] (3) Fluorescence detection: Set up the FAM fluorescence channel, incubate the reaction system at different temperatures for 1 h, detect the fluorescence intensity every 1 min, and process the collected signals.

[0124] Note: LbuCas13a enzyme was purchased from Bio-lifesci (Guangzhou, China).

[0125] II. Experimental Results Figure 16 A represents the fluorescence growth curve of 10pM target DNA detected by blocking crRNA with ssDNA_handle, ssDNA_handle_all_extend_12nt, and ssDNA_all at 38℃. The blocking effect of crRNA gradually increases with the increase of ssDNA sequence length. The fluorescence intensity at the end of the fluorescence growth curve is used to characterize the trans-cleavage activity of the Cas13a enzyme. The corresponding results are shown in Figure 1. Figure 16 As shown in B. Therefore, a temperature gradient experiment was performed on ssDNA_all, and the results are as follows. Figure 16As shown in Figure C, ssDNA_all inhibited Cas13a enzyme activity under different temperature gradients, but its blocking effect was significantly weaker than that of Cas12a enzyme. Furthermore, the activity of Cas13a enzyme showed a temperature-dependent difference, with significantly higher activity at low temperatures than at high temperatures. The blocking effect at this stage was also characterized by the fluorescence intensity at the endpoint of the fluorescence growth curve, as shown in the results below. Figure 16 As shown in D.

[0126] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for controlling the activity of Cas enzyme in a CRISPR-Cas system by temperature, characterized in that, Adding single-stranded DNA to the CRISPR-Cas system inhibits Cas enzyme activity at temperature 1 and activates Cas enzyme activity at temperature 2. Under the specified temperature 1 condition, the single-stranded DNA and the handle and / or spacer regions of the crRNA undergo complementary base pairing to form a closed crRNA. The crRNA blocked at temperature 2 is thermally denatured into a single strand.

2. The method as described in claim 1, characterized in that, Some of the bases in the single-stranded DNA have been modified with locked nucleic acids and / or methoxy groups.

3. The method as described in claim 1, characterized in that, The Cas enzyme is selected from any one of Cas12a, Cas12b and Cas13a enzymes.

4. The method as described in claim 1, characterized in that, Temperature 1 is lower than temperature 2.

5. A one-pot hot-start nucleic acid detection method combining RPA amplification and CRISPR-Cas12a-mediated activation, characterized in that, Includes the following steps: S11: Mix single-stranded DNA and crRNA, anneal at 90-95℃ to obtain blocked crRNA; in the blocked crRNA, the single-stranded DNA and the handle and / or spacer regions of the crRNA are complementary base pairs. S12: Mix Cas12a enzyme, RPA reaction reagent, Cas12a-reporter, target gene, and blocked crRNA to obtain the RPA-CRISPR-Cas12a reaction system. First, incubate at 35-38℃ to maintain the blocked state of the crRNA and perform RPA pre-amplification. Then, heat to 40-45℃ to release the blocked state of the crRNA, activate Cas12a enzyme activity, perform CRISPR-Cas12a reaction, and detect the fluorescence intensity of the target gene.

6. A one-pot hot-start nucleic acid detection method combining LAMP amplification and CRISPR-Cas12b-mediated activation, characterized in that, Includes the following steps: S21: Mix single-stranded DNA and crRNA, anneal at 90-95℃ to obtain blocked crRNA; in the blocked crRNA, the single-stranded DNA and the handle region of the crRNA are complementary base pairs. S22: Mix Cas12b enzyme, LAMP reaction reagent, Cas12b-reporter, target gene, and blocked crRNA to obtain the LAMP-CRISPR-Cas12b reaction system. First, incubate at 50-55℃ to maintain the blocked state of the crRNA and perform LAMP pre-amplification. Then, raise the temperature to 60-65℃ to release the blocked state of the crRNA, activate Cas12b enzyme activity, perform CRISPR-Cas12b reaction, and detect the fluorescence intensity of the target gene.

7. A one-pot hot-start nucleic acid detection kit combining RPA amplification and CRISPR-Cas12a-mediated activation, characterized in that, The kit contains Cas12a enzyme, RPA reaction reagent, Cas12a-reporter, crRNA, and single-stranded DNA, wherein the single-stranded DNA is complementary to the handle and / or spacer regions of the crRNA.

8. The kit according to claim 7, characterized in that, The nucleotide sequence of the crRNA is shown in SEQ ID NO: 11, and the nucleotide sequence of the single-stranded DNA is shown in any one of SEQ ID NO: 2 to 10. Some bases in the single-stranded DNA are modified with locked nucleic acids and / or methoxy groups.

9. A one-pot hot-start nucleic acid detection kit combining LAMP amplification and CRISPR-Cas12b-mediated activation, characterized in that, The kit contains Cas12b enzyme, LAMP reaction reagent, Cas12b-reporter, crRNA, and single-stranded DNA, wherein the single-stranded DNA is complementary to the handle region of the crRNA.

10. The kit according to claim 9, characterized in that, The nucleotide sequence of the crRNA is shown in SEQ ID NO: 18, and the nucleotide sequence of the single-stranded DNA is shown in SEQ ID NO: 17.

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