Universal crRNA, light-operated probe, nucleic acid detection method and application

Abstract

The invention relates to universal crRNA, a light-operated probe, a nucleic acid detection method and application, and relates to the technical field of biological detection. The universal crRNA comprises a 5 'SDR region containing a 5' DR region, and a 3 'SDR-spacer region containing a 3' DR region and a spacer region; the 5 'DR region and the 3' DR region are obtained by splitting the DR region, and the splitting position is located in the ring region of the stem-ring structure; the 5 'SDR region is provided with a cage modification, and the spacer region is used for identifying a target sequence. The crRNA not only can maintain the original functions of wild crRNA, but also can realize silencing and activation of the crRNA through light control, and due to the unique structural design of the crRNA, the crRNA has universality, only needs to synthesize and replace partial regions aiming at different target sequences, and has the characteristics of simplicity and stability.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological detection, in particular to a universal crRNA, a light-controlled probe, a nucleic acid detection method and application. BACKGROUND

[0002] Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas system is an emerging and promising biotechnology tool. CRISPR-Cas tool was originally developed for gene editing, and has been rapidly developed in gene regulation, imaging, therapy and the latest molecular diagnosis in recent years. CRISPR technology has some unprecedented characteristics, such as precise nucleic acid recognition mediated by guide RNA, homogeneous reaction and strong signal amplification mechanism, which are extremely attractive for the development of the next generation of nucleic acid detection technology. Therefore, a series of CRISPR-based nucleic acid detection methods have emerged. Among these methods, the technology combining isothermal nucleic acid amplification with CRISPR recognition, such as Specific High-sensitivity Enzymatic Reporter Unlocking (SHERLOCK), DNA endonuclease-targeted CRISPR Transcriptional Reporter (DETECTOR) and Hour-long, Low-cost, Multiplexed, Efficient Reporter (HOLMES), is the most widely studied because they can be performed at a single reaction temperature and meet the sensitivity requirements of clinical detection.

[0003] However, these platform technologies still face a key bottleneck in commercial applications: CRISPR cleavage reaction is incompatible with nucleic acid amplification reaction. When the isothermal amplification system is combined with the CRISPR reaction in a single tube, the CRISPR cleavage will destroy the nucleic acid amplification template, thereby reducing the amplification efficiency and detection sensitivity. Therefore, in the original versions of SHERLOCK, DETECTOR and HOLMES, nucleic acid amplification and CRISPR reaction are carried out in different test tubes. However, the two-step reaction requires opening the reaction tube and performing liquid transfer, which increases the complexity of the detection procedure. In addition, the liquid transfer step is extremely susceptible to aerosol contamination, which can lead to false positive results. Although some improvements have been made subsequently, including spatial isolation of reagents, development of buffer or suboptimal protospacer adjacent motif (PAM) sites to weaken the CRISPR reaction, or use of phase separation solution, these methods are still inconvenient or will inhibit the efficiency of the CRISPR reaction, so the compatibility problem has not been well solved. SUMMARY

[0004] In view of the above technical problems, the present application provides a universal crRNA, which can not only maintain the original function of the wild-type crRNA, but also realize the silencing and activation of the crRNA through light control, and the unique structural design of the crRNA makes it universal, only needs to be synthesized and replaced in part regions for different target sequences, and has the characteristics of simplicity and stability.

[0005] The present application provides a universal crRNA, comprising a 5' SDR region, a 3' SDR-spacer region, the 5' SDR region comprising a 5' DR region, the 3' SDR-spacer region comprising a 3' DR region and a spacer region; the 5' DR region and the 3' DR region are obtained by splitting the DR region, and the splitting position is located in the loop region of the stem-loop structure.

[0006] The 5' SDR region is provided with a caging modification.

[0007] The spacer region is used for recognizing a target sequence, and the spacer region is located at one end of the 3' DR region away from the loop region.

[0008] In view of the problem that the above amplification system and detection system cannot be well compatible, thereby leading to the inability to realize one-step method, the idea of light-controlled CRISPR technology is proposed in the early research. Specifically, by using light-controlled silencing RNA or using light-responsive group to modify the crRNA, the function of the crRNA is temporarily inhibited. When the nucleic acid amplification reaction is completed, 356nm ultraviolet light is used for irradiation, and the inhibition of the silencing RNA or the light-responsive group modification on the crRNA is removed, and then the recognition of the Cas enzyme to the target gene amplification product is started. The specific light control strategy can be divided into two categories: (1) covalent modification of the light-responsive group of the crRNA, such as modification of the cage group (NPOM-dT) in the spacer region or PLG acylation modification of the RNA sequence phosphate skeleton; (2) light-controlled silencing probe strategy, that is, designing a complementary RNA blocking chain of the crRNA, and breaking the chain through light cleavage linker (PC linker) after ultraviolet light irradiation, so as to remove the silencing effect on the crRNA. The light control strategy can separate the nucleic acid isothermal amplification and the CRISPR reaction process in time under the premise of no opening cover, and solves the problem of low compatibility of the existing isothermal amplification system and the CRISPR reaction. However, the above light-controlled CRISPR technology still has significant limitations, and different crRNAs need to be individually optimized for the modification site of the light-controlled cage group (NPOM-dT), or specific light-responsive silencing probes are designed according to the corresponding sequence, or the crRNA is subjected to light-controlled PLG acylation modification. These factors all lead to the problems of high development cost, poor universality, and difficulty in realizing standardized production of the detection technology.

[0009] The conventional crRNA is composed of a DR region and a spacer region, wherein the DR region forms a stem-loop structure like a "hairpin", and binds and interacts with the Cas enzyme, which is crucial for the conformation and function of the Cas12 (or Cas13a, Cas13b) / crRNA complex; and the main function of the spacer region is to form an RNA:DNA heteroduplex complementary structure with the target DNA strand, which is mainly responsible for recognizing the target sequence. Therefore, the inventors of the present application propose the crRNA with the above-mentioned specific structural design, which has a 5' DR region from the DR sequence of the wild-type crRNA, a 3' DR region, and a variable spacer region. Since the structure of the 5' DR region and the 3' DR region is obtained by truncating and splitting the "loop" region of the wild-type DR sequence, the split structure retains the "stem" structure of the original stem-loop structure in the DR region, so that the original function of the crRNA is stably maintained, and the variable spacer region is ingeniously utilized, which can be designed and synthesized according to the target sequence. At the same time, the 5' SDR region is provided with a caging modification. When the designed crRNA needs to be silenced, the caging group will prevent the Watson-Crick base pairing between the 5' SDR region and the 3' SDR-spacer region, and will also affect the interaction between RNA and Cas protein, thereby making the activity of the CRISPR system in the "silent" state and unable to be activated by the amplification product; when activation is needed, the 5' SDR region can be uncaged by ultraviolet irradiation, and the uncaged 5' SDR can form a functional structure with the 3' SDR-spacer and form a complex with the Cas enzyme, so that the CRISPR system is in a normal state that can be activated, thereby realizing the effect of light control. More importantly, since the 5' DR region in the crRNA of the present application is derived from the wild-type crRNA and has conservation, it can be used universally, so as long as the spacer region is designed and synthesized according to different target sequences, the 3' SDR-spacer region is composed of the 3' DR region, and then replaced and combined with the caged 5' SDR region, the cost can be greatly reduced while the original crRNA functionality is retained, and the method is simple and stable.

[0010] In one embodiment, the sequence of the spacer region is base complementary to the target sequence.

[0011] In one embodiment, the 5' SDR region further comprises a 5' extension region located at one end of the 5' DR region close to the loop region.

[0012] The 3' SDR-spacer region further comprises a 3' extension region located at one end of the 3' DR region close to the loop region.

[0013] The sequence of the 5' extension region is base complementary to the sequence of the 3' extension region.

[0014] In one of the embodiments, the sequence of the 5' extension region is ≥ 5 nt, and the sequence of the 3' extension region is ≥ 5 nt.

[0015] In one of the embodiments, the length of the sequence of the 5' extension region is 5-13 nt, and the length of the sequence of the 3' extension region is 5-13 nt.

[0016] In one of the embodiments, the length of the sequence of the 5' extension region is 6-13 nt, and the length of the sequence of the 3' extension region is 6-13 nt.

[0017] In one of the embodiments, the number of sites of the caging modification in the 5' SDR region is ≥ 2.

[0018] In one of the embodiments, the caging modification comprises: replacing U base with NPOM-dT.

[0019] The application further provides a CRISPR-Cas detection system comprising a Cas enzyme and the universal crRNA.

[0020] In one of the embodiments, the Cas enzyme is a Cas12 enzyme.

[0021] In one of the embodiments, the Cas12 enzyme is any one of Cas12a, Cas12c, Cas12d, Cas12e, Cas12f, Cas12g, Cas12h, Cas12i, Cas12j or Cas12k.

[0022] In one of the embodiments, the Cas12 enzyme is Cas12a.

[0023] In one of the embodiments, the Cas12a is any one of LbaCas12a, FnCas12a or AsCas12a.

[0024] The application further provides a kit comprising a constant-temperature nucleic acid amplification system and the CRISPR-Cas detection system.

[0025] In one of the embodiments, the constant-temperature amplification system comprises a RAA amplification system.

[0026] The application also provides a target sequence detection method for non-diagnostic purposes, which is realized by the kit and comprises the following steps: amplifying the target sequence by using the constant temperature nucleic acid amplification system to obtain an amplification product, irradiating the universal crRNA in the CRISPR-Cas detection system with ultraviolet light to activate the universal crRNA, and detecting the amplification product.

[0027] In one embodiment, the ultraviolet light irradiation time is greater than or equal to 5 seconds.

[0028] Compared with the prior art, the application has the following beneficial effects:

[0029] The universal crRNA, light-controlled probe, nucleic acid detection method and application provided by the application not only maintain the original functions of wild-type crRNA, but also realize the silencing and activation of crRNA through light control. The unique structural design of the crRNA makes it universal, and only partial regions need to be synthesized and replaced for different target sequences, which has the characteristics of simplicity and stability. The "one-tube method" constructed based on the universal crRNA not only can better adapt to the amplification system and the CRISPR-Cas detection system, but also has high detection sensitivity. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 Figure 1 is a diagram showing the design scheme and sequence length optimization results of the direct split repeat sequence (DR) of crRNA, wherein, Figure 1 a is a diagram showing the design scheme of the direct split repeat sequence (DR) of crRNA, Figure 1 b is a diagram showing the length evaluation and optimization results of the 5' end split direct repeat sequence (5'SDR), Figure 1 c is a diagram showing the length evaluation and optimization results of the 3' end split direct repeat sequence (3'SDR);

[0031] Figure 2 Figure 2 is a diagram showing the CRISPR-RNA detection efficiency comparison results based on different split strategies, wherein, Figure 2 a is a fluorescence curve of the CRISPR reaction using wild-type crRNA (wt-crRNA); Figure 2 b is a fluorescence curve of the CRISPR reaction of CRISPR-RNA based on the split direct repeat sequence (split DR split mode) of crRNA; Figure 2 c is a fluorescence curve of the CRISPR reaction of CRISPR-RNA based on the split direct repeat sequence and spacer (DR+spacer split mode) of crRNA; Figure 2Figure d shows the comparison of detection efficiency of wild-type crRNA, CRISPR-RNA with DR splitting mode, and CRISPR-RNA with DR+spacer splitting mode.

[0032] Figure 3 The figure shows the results of the construction and performance evaluation of the light-controlled universal CRISPR-RNA system. Figure 3 a is a schematic diagram of the design of a light-controlled universal 5' split direct repeat sequence (5'SDR); Figure 3 b is a graph showing the results of general 5'SDR activity assessment after cage modification at different sites in the sequence; Figure 3 c is a graph showing the universality assessment results of the universal 5'SDR with caged modification at different sites in the sequence; Figure 3 d is the result of optimizing the ultraviolet irradiation time; Figure 3 e is a graph showing the evaluation results of the detection sensitivity of universal light-controlled crRNA;

[0033] Figure 4 This is a schematic diagram illustrating the working principle of the universal crRNA of this invention.

[0034] Figure 5 The diagram illustrates the working principle and performance verification results of the "one-tube method" isothermal nucleic acid amplification-Cas12a technology; among them, Figure 5 a is a schematic diagram of the principle of the "one-tube method" isothermal nucleic acid amplification-Cas12a technology based on the universal optically controlled 5'SDR; Figure 5 Figure b shows the feasibility verification results of the one-tube RAA-Cas12a system based on the universal optical control 5'SDR. Figure 5 c is a graph showing the verification results of the detection sensitivity of the one-tube RAA-Cas12a system;

[0035] Figure 6 The image shows the clinical performance validation results of the light-controlled one-tube RAA-Cas12a detection system (conven. or conventional refers to the traditional two-step RAA-Cas12a system); among them, Figure 6 a is a fluorescence thermogram of the detection results of Mycoplasma pneumoniae positive clinical samples by the photocontrolled one-tube method and the traditional two-step method RAA-Cas12a system; Figure 6 b is a graph showing the results of ROC analysis of clinical sample testing data; Figure 6 c shows the results of detecting MP clinical specimens using the light-controlled one-tube RAA-Cas12a method; Figure 6 d represents the results of detecting MP clinical specimens using the traditional two-step RAA-Cas12a method; Figure 6 e is a graph showing the analysis of the detection results of clinical specimens of MP using the light-controlled one-tube RAA-Cas12a method; Figure 6f represents the analysis of the detection results of MP clinical specimens using the traditional two-step RAA-Cas12a method; Figure 6 g is a graph showing the performance parameter analysis and comparison results of the detection methods. Detailed Implementation

[0036] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0038] Example

[0039] I. Design and construction of universal crRNA.

[0040] To construct a universal light-controlled CRISPR system, wild-type crRNA was first modified. The wild-type crRNA (wt-crRNA) sequence is shown in SEQ ID NO: 1. Figure 1 As shown in a, the "loop" of the hairpin structure in the Direct Repeat region of crRNA is divided into a sequence-constant 5'DR region and a sequence-variable 3'DR-spacer region, wherein the 3'DR-spacer region is composed of the 3'DR region and the spacer region.

[0041] To investigate the minimum number of sequences from the DR structure required to maintain crRNA function, the sequence lengths of the 5'DR and 3'DR regions formed after the DR structure was split were gradually shortened.

[0042] Simultaneously, to eliminate the potential adverse effects of DR structure splitting on its function, an extension sequence is performed at the splitting point located at the end of the "loop" to form an additional complementary RNA double-strand region, aiming to stabilize the RNA hybridization structure. The sequence obtained by extending the 5'DR region from the end near the splitting point is the 5' extension region, and the 5' extension region and the 5'DR region together form the 5'SDR region; the sequence obtained by extending the 3'DR region from the end near the splitting point is the 3' extension region, and the 3' extension region, the 3'DR region, and the spacer region together form the 3'SDR-spacer region. It is understood that the specific implementation methods of the above extension sequences can be selected and implemented by those skilled in the art based on existing technologies, such as artificially synthesized bases.

[0043] Based on the above principles, in order to study whether different lengths of 5'DR region, 3'DR region, and different lengths of extended region affect the performance of crRNA, sequences of 5'SDR and 3'SDR-spacer were designed and screened. The sequences of the designed 5'SDR region are shown in SEQ ID NO: 2-SEQ ID NO: 14, and the sequences of the 3'SDR-spacer region are shown in SEQ ID NO: 15-SEQ ID NO: 23.

[0044] Specific procedures for crRNA activity detection: ① Preparation of synthesized target dsDNA fragments. Mix 20 μL each of 10 μM target ssDNA (59 nt long) and 10 μM target ssDNA complementary strand (59 nt long) with 5 μL of 10×PCR buffer to a final volume of 50 μL. Perform gradient annealing in a PCR instrument at 95℃ for 5 min, followed by gradient cooling at 1℃ every 10 s until reaching room temperature, yielding a 4 μM target dsDNA concentration. Dilute to 5 nM with 0.5×PCR buffer and freeze for later use. ② Preparation of the Cas12a reaction system. Prepare the Cas12a reaction solution under ice bath conditions. Add 8 μL of Cas12a reaction solution to a qPCR tube, then add 2 μL of target dsDNA at a concentration of 5 nM. After vortexing and mixing, obtain 10 μL of detection reaction solution containing 1×NEB buffer 2.1, 30 nM baCas12a protein, 500 nM MFQ DNAreporter, 50 nM 5'SDR, and 50 nM 3'SDR-spacer. Incubate on a real-time PCR instrument at 37°C for 60 min, recording fluorescence values ​​every 60 s.

[0045] During the experiment, it was found that when the 5'DR was gradually shortened, for example, when the 5'SDR was 6 nt, 8 nt, and 10 nt, there was basically no activity. The crRNA activity detection results when the 5'SDR was 10 nt were obtained in [the following text is missing from the original] Figure 1 As shown in b, we believe that even if the original DR region is split, it needs to be fully incorporated into the modified crRNA; reducing its length would cause the modified crRNA to lose its function. The effects of different length extension regions are shown in the results. Figure 1 (bc) The artificially introduced double-stranded RNA structure is essential for maintaining the function of CRISPR-RNA. When the length of this double-stranded RNA segment is only 2 base pairs, the CRISPR-RNA almost loses its function. Correspondingly, when the double-stranded RNA structure is extended to 4 base pairs, the function is significantly restored. And after extending to 5 base pairs, its activity recovers to a level almost equivalent to that of wild-type crRNA. When the length of the 5' SDR is between 17 and 25 base pairs and the length of the 3' SDR is between 15 and 22 base pairs, the activity of split crRNA can be maintained at a level equivalent to that of wt-crRNA (wild-type), and when the SDR length is within these ranges, the sequence length has no observable effect on its activity. Considering the cost of synthesis, the inventors did not further experiment with longer extension regions, but the above experimental data show that the stability of the modified crRNA increases with the increase of the base length of the extension region, and the effect on stability plateaus after the base length of the extension region is ≥5 nt. Based on this, the inventors selected a 5'SDR with a length of 21nt and a 3'SDR with a length of 18nt for subsequent experiments.

[0046] II. Performance verification of the universal crRNA of the present invention.

[0047] Previous studies have shown that wild-type crRNA can be split into two segments, DR and spacer, while the DR sequence remains intact. Therefore, the DR sequence fragment obtained based on the splitting idea of ​​DR region + spacer region can also be used to design a universal light-controlled RNA module.

[0048] Therefore, the inventors conducted performance verification on the DR+spacer splitting mode and the crRNA formed by the splitting DR splitting mode of the present invention. The specific operations are as follows: ① The crRNA obtained by splitting DR splitting mode and the performance verification experimental steps are the same as the "Specific Operation of crRNA Activity Detection Experiment" section of "I. Design and Construction of Universal crRNA"; ② DR+spacer splitting mode. The DR+spacer splitting mode only splits the crRNA into the DR region and the spacer region (as shown in SEQ ID NO: 24 and SEQ ID NO: 25), without sequence modification. The performance verification experimental steps are as follows: Prepare the Cas12a reaction solution under ice bath conditions. Add 8 μL of the Cas12a reaction solution to a qPCR tube, add 2 μL of target dsDNA at a concentration of 5 nM, vortex to mix, and obtain 10 μL of detection reaction solution containing 1×NEB buffer 2.1, 30 nM LbaCas12a protein, 500 nM FQ DNAreporter, 50 nM DR, and 50 nM spacer. Incubate the solution at 37℃ for 60 min on a quantitative PCR instrument, recording the fluorescence value every 60 s.

[0049] The results showed that, in the DR+spacer splitting mode, the CRISPR reaction activity was not high in the initial stage. Figure 2 This results in a reaction rate that cannot reach the level of wild-type crRNA (analysis of the initial reaction rate plot). However, the splitting mode of DR in this invention has a significantly higher reaction efficiency than the DR+spacer splitting mode, and is closer to the wild-type crRNA (…). Figure 2 ).

[0050] 3. Form a light-controlled universal crRNA and verify its function.

[0051] Building upon the previously constructed general-purpose 5'SDR fragment, further efforts were made to develop a light-controlled general-purpose 5'SDR. NPOM-dT (light-sensitive, 6-nitropiperonyloxymethyl–modified deoxynucleotide thyminecaged nucleotide) is a commercially available light-sensitive caged nucleotide. The synthesis and preparation of light-controlled 5'SDRs (commercially available) were achieved by replacing the U base with NPOM-dT. A series of light-controlled nucleic acid fragments were constructed by modifying the U base sites in the 5'SDR sequence with light-controlled caged nucleotides. Figure 3a) The target sequence is Target1 (double-stranded DNA), prepared by annealing single-stranded DNA Target1-F and single-stranded DNA Target1-R, and then diluted to the required concentration. The specific synthesis operation is as shown in the step "① Preparation of the synthesized target dsDNA fragment" of "Specific operation of crRNA activity detection experiment" in "I. Design and construction of universal crRNA". Single-stranded DNA Target1-F is shown as SEQ ID NO: 26, single-stranded DNA Target1-R is shown as SEQ ID NO: 27, and the light-controlled nucleic acid fragments 5′SDR-C5, 5′SDR-C6, 5′SDR-C8, 5′SDR-C11, and 5′SDR-C(8+11) are SEQ ID NO: 28-34, respectively.

[0052] Functional recovery and silencing verifications were performed on crRNAs modified with light-controlled cage groups at different sites. The specific procedures for functional recovery and silencing verification were as follows: Cas12a reaction solution was prepared under ice bath conditions. 8 μL of Cas12a reaction solution was added to a qPCR tube, followed by 2 μL of target dsDNA at a concentration of 5 nM. After vortexing, 10 μL of detection reaction solution containing 1×NEB buffer 2.1, 30 nM LbaCas12a protein, 500 nM FQ DNA reporter, 50 nM light-controlled 5'SDR, and 50 nM 3'SDR-spacer were obtained. The experiment included a light-treated group and a non-light-treated group. The light-treated group was irradiated with a 365 nm UV lamp (λ = 365 nm, 35 W) for 20 s, while the non-light-treated group was not subjected to light treatment. Finally, the reaction tubes were incubated at 37 °C for 60 min on a quantitative PCR instrument, and fluorescence values ​​were recorded every 60 s.

[0053] We first explored strategies for single-base site cage modification, and initial results showed that ( Figure 3 (b) After UV irradiation, the function of 5'SDR at all modified sites was fully restored to a level comparable to or even better than that of unmodified 5'SDR, indicating that replacing U bases with dT bases had no effect on its function. However, when evaluating the silencing effect of caged modification on 5'SDR, the results showed that only caged modification at site 6 achieved the expected effect, i.e., caged modification could silence RNA function. After replacing U bases with NPOM-dT at sites 5, 8, and 11, 5'SDR still retained weak activity.

[0054] Next, to further verify the universality of the cage modification at site 6, the specific procedures were as follows: Cas12a reaction solution was prepared under ice bath conditions. 8 μL of Cas12a reaction solution was added to a qPCR tube, followed by 2 μL of 5 nM target dsDNA1 (Target1). After vortexing, 10 μL of detection reaction solution containing 1×NEB buffer 2.1, 30 nM baCas12a protein, 500 nM FQ DNA reporter, 50 nM 5'SDR light-controlled PCR, and 50 nM 3'SDR18-spacer1 was obtained. The experiment included a light-treated group and a non-light-treated group. The light-treated group was irradiated with a 365 nm UV lamp (λ = 365 nm, 35 W) for 20 s, while the non-light-treated group received no light treatment. Finally, the reaction tubes were incubated at 37°C for 60 min on a quantitative PCR instrument, and fluorescence values ​​were recorded every 60 s. The 3'SDR18-spacer used for the test target dsDNA2-6 (Target2-6) was 3'SDR18-spacer2-6, and the parameters of the other components were the same.

[0055] The results showed that when different spacer sequences were designed to detect different target DNAs (target1-6, corresponding to spacer1-6 sequences), cage modification at site 6 could not completely silence the function of 5'SDR. Therefore, cage modification at this site lacks universality. Figure 3 c). Therefore, we hypothesize that it may be necessary to increase the number of modification sites, and we explored a dual-modification site strategy that simultaneously performs cage modification at base sites 6+8, 6+11, and 8+11. Figure 3a). When the target sequence is Target2, 3'SDR18-spacer2 is as shown in SEQ ID NO: 35, Target2-F is as shown in SEQ ID NO: 36, and Target2-R is as shown in SEQ ID NO: 37; when the target sequence is Target3, 3'SDR18-spacer3 is as shown in SEQ ID NO: 39, Target3-F is as shown in SEQ ID NO: 40, and Target3-R is as shown in SEQ ID NO: 41; when the target sequence is Target4, 3'SDR18-spacer4 is as shown in SEQ ID NO: 42, Target4-F is as shown in SEQ ID NO: 43, and Target4-R is as shown in SEQ ID NO: 44; when the target sequence is Target5, 3'SDR18-spacer5 is as shown in SEQ ID NO: 45, Target5-F is as shown in SEQ ID NO: 46, and Target5-R is as shown in SEQ ID NO: 47; when the target sequence is Target6, 3'SDR18-spacer6 is as shown in SEQ ID NO: 48, and Target6-F is as shown in SEQ ID NO: 37. As shown in ID NO: 49, Target6-R is shown in SEQ ID NO: 50. The synthesis method of the above target sequences Target2-6 is the same as that of Target1.

[0056] The results showed that no signal was observed after dibase cage modification at sites 6+8 and 6+11, regardless of the presence or absence of the target Target1. Figure 3 (b) To rule out experimental randomness, we further tested its functionality using different 3'SDR18-spacers and corresponding targets. Figure 3 c) It was found that modifications at sites 6+8 and 6+11 could completely silence the function of 5'SDR. These two modification modifiers showed slightly weaker response signals for some targets, generally reaching product usability levels, but very weak detection signals for others. This indicates that modifications at sites 6+8 and 6+11 have good silencing performance, but lack versatility. Double-base cage modification at site 8+11 could completely silence the function of 5'SDR, and after removing the cage group by light, the RNA function recovered to the same level as the unmodified 5'SDR. Figure 3 bc). Moreover, this caged RNA demonstrated excellent versatility in detecting different targets. Figure 3 c).

[0057] The 3'SDR18-spacer modified with double base cageing at sites 8+11 was selected. The required illumination time for photoactivated crRNA was tested using UV irradiation of varying durations. The specific procedure was consistent with the experimental steps in "III. Formation of Photoactivated Universal crRNA and Functional Verification" ("Further Verification of the Universality of Cage Modification at Site 6"), differing only in the duration of UV irradiation. Results showed that UV irradiation for 5 seconds and longer completely activated the crRNA's function. Figure 3 d).

[0058] Based on the above, further verification was conducted to determine whether the engineering modification and photosensitive group modification would damage the performance of crRNA. Specific procedures included: ① dsDNA target dilution. The prepared target dsDNA3 was diluted to 5000 pM, 500 pM, 50 pM, 5 pM, 5 pM, and 0 pM using EASY Dilution (Takara, 9160Q) solution; ② Preparation of light-controlled Cas12a reaction solution. The Cas12a reaction solution was prepared under ice bath conditions. 8 μL of the Cas12a reaction solution was added to a qPCR tube, and 2 μL of target dsDNA3 at different concentrations was added to each reaction tube. After vortexing and mixing, 10 μL of detection reaction solution containing 1×NEB buffer 2.1, 30 nM LbaCas12a protein, 500 nM FQ DNA reporter, 50 nM light-controlled 5'SDR, and 50 nM 3'SDR18-spacer3 was obtained. Irradiate the reaction tubes for 20 seconds under a 365nm UV light source (UV lamp, λ=365nm, 35W), then place the reaction tubes on a quantitative PCR instrument and incubate at 37℃ for 60 minutes, recording the fluorescence value every 60 seconds. ③ Preparation of traditional Cas12a reaction solution. Prepare the Cas12a reaction solution under ice bath conditions. Add 8μL of Cas12a reaction solution to a qPCR tube. Add 2μL of target dsDNA3 at different concentrations to different reaction tubes, vortex to mix, and obtain 10μL of detection reaction solution containing 1×NEB buffer 2.1, 30nM LbaCas12a protein, 500nM FQ DNA reporter, and 50nM wt-crRNA3. Place the reaction tubes on a quantitative PCR instrument and incubate at 37℃ for 60 minutes, recording the fluorescence value every 60 seconds.

[0059] The results show ( Figure 4e) Both the light-controlled crRNA (Split crRNA) and the wild-type crRNA (wt-crRNA, sequence shown in SEQ ID NO: 38) were able to directly detect target DNA at concentrations as low as 0.1 pM, indicating that the performance of the light-controlled crRNA was not affected. These results demonstrate that a universal caged light-controlled 5'SDR was successfully designed and constructed.

[0060] IV. Constructing a single-tube light-controlled RAA-Cas12a nucleic acid detection system.

[0061] Next, a one-tube optically controlled RAA-Cas12a nucleic acid detection system was constructed using a CRISPR-Cas12a system based on a universal optically controlled 5'SDR.

[0062] The working principle diagram of the universal crRNA of this invention is as follows: Figure 5 As shown, the principle of the "one-tube method" isothermal nucleic acid amplification-Cas12a technology based on the universal optically controlled 5'SDR is as follows: Figure 5 As shown in Figure a, during the isothermal nucleic acid amplification stage, the caged 5'SDR sequence cannot form the correct CRISPR-RNA sequence hybridization structure with the 3'SDR-spacer sequence, leaving the CRISPR system in a silent state. At this time, the isothermal amplification reaction (RAA) can proceed normally without interference from the CRISPR system. After nucleic acid amplification is complete, brief ultraviolet light irradiation decages the 5'SDR, thereby restoring the CRISPR system's function. The CRISPR system recognizes the nucleic acid amplification product, Cas12a is activated, and its trans-cleavage activity cleaves the reporter probe, generating a detection signal, thus achieving detection.

[0063] In this embodiment, the isothermal amplification reaction used the RAA nucleic acid amplification kit (B00000) purchased from Jiangsu Qitian Gene Biotechnology Co., Ltd.

[0064] The specific operation of the feasibility verification experiment: The system of the light-controlled one-tube reaction consists of three components: a, b, and c. Component a: 1 μL of 10×NE Buffer 2.1, 1 μL of 1 μM 5'SDR, 1 μL of 1 μM 3'SDR-spacer, 0.8 μL of 10 μM FQ probe, 0.5 μL of 1 μM LbCas12a, and 0.7 μL of enzyme-free water, totaling 5 μL. Component b: According to the instructions of the RAA nucleic acid amplification kit (B00000), 25 μL of reaction buffer was added to the lyophilized powder to prepare a RAA suspension, which was then mixed with 0.4 μL of 10 μM forward primer, 0.4 μL of 10 μM reverse primer, 5 μL of RAA suspension, 2 μL of target DNA, and 1.2 μL of enzyme-free water to obtain a 9 μL component. Component c is 1 μL of magnesium acetate (MgOAc). After preparation, add component B to the bottom of the qPCR tube, then add components A and C to the same tube wall. Briefly centrifuge to remove the reagents from the bottom of the tube, and incubate at 37°C for 30 min on a real-time PCR instrument, recording the fluorescence value every 60 s. Then, irradiate the reaction tube with UV (λ356nm) for 20 s. Finally, incubate again at 37°C for 60 min on a real-time PCR instrument, recording the fluorescence value every 60 s.

[0065] Based on the above reaction system, a feasibility verification experiment was designed, and the missing components in each reaction group were replaced with nuclease-free water.

[0066] The specific procedures for the sensitivity verification experiment are as follows: To facilitate concentration quantification, plasmid DNA containing the target gene fragment was used as the target gene. The plasmid DNA was diluted to the appropriate concentration using EASY Dilution (Takara, 9160Q). The light-controlled single-tube reaction procedure was the same as the feasibility experiment procedure. Based on this, plasmid DNA of different concentrations was added to the light-controlled single-tube reaction solution. After preparing the reaction solution, the tube was briefly centrifuged to remove the reagents from the bottom, and then incubated at 37°C for 30 min on a real-time PCR instrument, recording the fluorescence value every 60 s. Subsequently, the reaction tube was irradiated with ultraviolet light (λ356nm) for 20 s. It was then incubated again at 37°C for 60 min on a real-time PCR instrument, recording the fluorescence value every 60 s.

[0067] The target genes for the above feasibility and sensitivity verification experiments are shown in SEQ ID NO: 53, the amplification primer sequence MP-F is shown in SEQ ID NO: 51, and the amplification primer sequence MP-R is shown in SEQ ID NO: 52.

[0068] like Figure 5As shown in b, the feasibility of the single-tube light-controlled RAA-Cas12a nucleic acid detection system was first verified. The results showed that the reaction could only produce a significant fluorescence signal when all the necessary elements, such as isothermal nucleic acid amplification, the Cas12a system, and ultraviolet light irradiation, were present. This proved that the single-tube light-controlled RAA-Cas12a nucleic acid detection system based on the universal light-controlled 5'SDR is feasible.

[0069] The analytical sensitivity of the detection system was further verified, such as Figure 6 As shown in the results in c, the detection system has a sensitivity of 0.2 fg (approximately equivalent to 1 gene copy) for plasmid DNA containing the target gene fragment, demonstrating its excellent detection sensitivity.

[0070] V. Clinical Applications.

[0071] To further evaluate its clinical application value, a performance evaluation of this new method in the detection of Mycoplasma pneumoniae in clinical samples was conducted. First, 55 remaining Mycoplasma pneumoniae samples from the hospital's molecular diagnostic laboratory were collected, including 42 positive samples and 13 negative samples.

[0072] The samples were tested using the one-tube light-controlled RAA-Cas12a system of this invention and the conventional two-step RAA-Cas12a system. The conventional two-step RAA-Cas12a system used commercially available products and LbacrRNA3, the sequence of which is shown in SEQ ID NO: 38. The specific reaction steps were as follows: RAA amplification reaction: 25 μL of reaction buffer was added to the lyophilized powder to prepare a RAA suspension. Then, 22.5 μL of amplification reaction solution containing 1 μL of 10 μM forward primer, 1 μL of 10 μM reverse primer, 12.5 μL of RAA suspension, 2 μL of target DNA, and enzyme-free water was prepared in a reaction tube. Finally, 2.5 μL of magnesium acetate was added to the tube wall. After mixing thoroughly and centrifuging, the mixture was incubated at 37°C for 30 min to obtain the amplification product. Cas12a detection reaction: 2 μL of RAA product was added to the Cas12a reaction solution to obtain a mixture containing 1×NEB Buffer 2.1, 30 nM LbCas12a, 50 nM crRNA, and 500 nM FQ probe. The mixture was incubated at 37°C for 60 min on a real-time PCR instrument, and fluorescence values ​​were recorded every 60 s.

[0073] The results are as follows Figure 6As shown in Figure a, when this batch of samples was tested using both the single-tube light-controlled RAA-Cas12a system and the traditional two-step RAA-Cas12a system, almost all positive samples showed extremely strong fluorescence signals. However, two samples tested with the single-tube light-controlled RAA-Cas12a system showed almost no signal (or extremely weak signal), while three positive samples tested with the traditional two-step method also showed almost no signal (or extremely weak signal). Two false negatives occurred where almost no signal was detected by either method. Figure 6 ROC analysis was performed on the detection data of all samples (53 cases), and the results showed that the AUC values ​​reached 0.956 (one-tube light-controlled method) and 0.9286 (traditional two-step method), respectively. Figure 6 b). The Youden index, obtained through ROC analysis, showed that the single-tube light-controlled method and the traditional two-step method missed 2 and 3 positive samples, respectively (e.g., Figure 6 cd).

[0074] Further analysis was conducted to determine the degree of agreement between the two aforementioned methods and the gold standard method (qPCR method, specifically using the Mycoplasma pneumoniae nucleic acid detection kit purchased from Sansure Pharmaceuticals, PCR-fluorescent probe method). Figure 6 Based on the above data, the detection sensitivity, specificity, and Kappa value were calculated, such as... ​ As shown in g, compared with the qPCR method, the light-controlled one-tube method has a detection sensitivity of 95.45%, a specificity of 100%, and a Kappa value of 96.37% for clinical samples, while the traditional two-step method has a detection sensitivity of 93.33%, a specificity of 100%, and a Kappa value of 94.55%. This demonstrates that the light-controlled one-tube method has detection performance no less than that of the traditional two-step method, and its detection capability is close to that of the clinical gold standard method, reflecting its high application value and potential.

[0075] The sequences involved in this invention are shown in the table below:

[0076]

[0077]

[0078]

[0079] Note: Some sequences in the table above are RNA sequences. Due to the letter standardization requirements of WIPO Sequence Listing, the uracil U in the RNA sequences will be replaced with T in the sequence listing file submitted with the application documents; sequences that have undergone cage modification will be presented as naked sequences in the sequence listing file.

[0080] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0081] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A universal crRNA, characterized in that, It includes a 5'SDR region and a 3'SDR-spacer region. The 5'SDR region includes a 5'DR region, and the 3'SDR-spacer region includes a 3'DR region and a spacer region. The 5'DR region and the 3'DR region are obtained by splitting the DR region. The splitting position is located in the ring region of the stem-ring structure. The 5'SDR region is provided with a cage-like modification; The spacer region is used to identify the target sequence, and the spacer region is located at the end of the 3'DR region away from the loop region.

2. The universal crRNA according to claim 1, characterized in that, The 5'SDR region also includes a 5' extension region, which is located at one end of the 5'DR region near the ring region; The 3'SDR-spacer region also includes a 3' extension region, which is located at one end of the 3'DR region near the ring region; The sequence of the 5' extension region is complementary to the sequence of the 3' extension region.

3. The universal crRNA according to claim 2, characterized in that, The sequence of the 5' extension region is ≥5nt, and the sequence of the 3' extension region is ≥5nt.

4. The universal crRNA according to claim 3, characterized in that, The length of the sequence in the 5' extension region is 5-13 nt, and the length of the sequence in the 3' extension region is 5-13 nt.

5. The universal crRNA according to any one of claims 1-4, characterized in that, The number of sites with caged modification in the 5'SDR region is ≥2.

6. The universal crRNA according to claim 5, characterized in that, The cage-like modification includes replacing the U base with NPOM-dT.

7. A CRISPR-Cas detection system, characterized in that, Includes Cas enzyme and universal crRNA as described in any one of claims 1-6.

8. The CRISPR-Cas detection system according to claim 7, characterized in that, The Cas enzyme is a Cas12 enzyme.

9. A reagent kit, characterized in that, Includes an isothermal nucleic acid amplification system and a CRISPR-Cas detection system as described in any one of claims 7-8.

10. A method for detecting target sequences for non-diagnostic purposes, characterized in that, The detection method is implemented using the kit described in claim 9, and includes the following steps: amplifying the target sequence using the isothermal nucleic acid amplification system to obtain the amplification product, irradiating the universal crRNA in the CRISPR-Cas detection system with ultraviolet light to activate the universal crRNA, and detecting the amplification product.

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