CRISPR (clustered regularly interspaced short palindromic repeats) detection system integrating target chain amplification and crRNA synthesis into one tube

Abstract

The invention discloses a CRISPR (clustered regularly interspaced short palindromic repeats) detection system integrating target chain amplification and crRNA synthesis into one tube, and belongs to the technical field of biosensing and molecular diagnosis. The system comprises an annular probe; the annular probe is amplified into double strands by phi29 DNA polymerase after being combined with a target, T7 RNA polymerase transcription is started to generate pre-crRNA, then the pre-crRNA is processed into mature crRNA by Cas12a, an ssDNA amplification product is recognized, an FQ reporter probe is cut, and a fluorescence signal is generated; wherein the sequence of the annular probe comprises a sequence complementary to a target chain, a single-chain T7 promoter sequence and a DNA sequence corresponding to a crRNA repetitive sequence. The detection platform integrates nucleic acid amplification and crRNA synthesis, real one-tube and one-step detection is realized, gRNA does not need to be additionally added, and the detection platform has the advantages of high sensitivity, high specificity, portability in operation and the like.

Description

Technical Field

[0001] The present invention relates to the fields of biosensing and molecular diagnosis, and in particular to a CRISPR detection system that integrates target chain amplification and crRNA synthesis in one tube. Background Art

[0002] With the continuous advancement of biomedical research, biosensor technology has become an important tool in modern diagnostics. Biosensor testing not only monitors changes in biomarkers in real time but also provides critical information for early diagnosis and treatment of diseases. Despite its numerous advantages, the complexity of its operational procedures and the specialized skills required limit its widespread application. Therefore, the development of simple and efficient detection technologies is crucial.

[0003] With the advancement of sensor detection technologies, rapid, accurate, and convenient molecular diagnostics are of great significance for the early detection and treatment of diseases. Nucleic acid detection technologies based on the CRISPR-Cas system have attracted considerable attention due to their high specificity and sensitivity. However, traditional nucleic acid detection methods typically require multiple steps, including sample extraction, amplification, and signal detection, which are time-consuming and susceptible to contamination. Therefore, the development of one-pot detection methods that can simultaneously perform amplification and detection in a single reactor is of particular importance. Despite the numerous advantages of one-pot detection, practical applications still face several challenges, such as interference between the amplification and detection steps and incompatibility of reaction conditions. To overcome these challenges, researchers have employed a variety of strategies, including physical isolation, microfluidic chips, enzyme characterization and engineering, and guide RNA modification. However, these methods suffer from drawbacks such as the need for human intervention, high costs, and technical complexity.

[0004] Therefore, the present invention proposes a detection method combining target chain amplification and gRNA synthesis. This detection technology requires the presence of an amplified activator for the production of mature crRNA, without the need for exogenous gRNA addition, which can effectively reduce the mutual interference between amplification and detection and achieve highly sensitive detection. This method can achieve true one-step, one-pot detection, reduce operational difficulty, and does not have the problem of incompatible reaction conditions. Summary of the Invention

[0005] To address the problems of interference between amplification and detection, incompatible reaction conditions, insufficient sensitivity, and complex operation in one-pot detection using existing CRISPR-Cas detection technology, the present invention provides a CRISPR detection system that integrates target chain amplification and crRNA synthesis in a single tube. It also integrates amplification and gRNA generation in a single tube, eliminating the need for exogenous gRNA addition. This system can achieve exponential amplification of biosensor signals, thereby improving detection sensitivity and simplifying the detection process.

[0006] In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions: A CRISPR detection system that integrates target strand amplification and crRNA synthesis in one tube includes: a circular probe (CP); after binding to the target strand RNA, the circular probe is amplified into a double strand by phi29 DNA polymerase and initiates transcription by T7 RNA polymerase to produce pre-crRNA. The pre-crRNA is then processed by Cas12a into mature crRNA, which recognizes the ssDNA amplification product and cleaves the FQ reporter probe to generate a fluorescent signal; The sequence of the circular probe includes a sequence complementary to the target chain, a single-stranded T7 promoter sequence, and a DNA sequence corresponding to the crRNA (CRISPR RNA) repeat sequence.

[0007] Furthermore, the method further comprises: monitoring the fluorescence signal by using a BioTek H1 microplate reader, wherein λ ex =492 nm; λ em =520 nm.

[0008] Furthermore, the circular probe includes a 20 nt sequence complementary to the target RNA, a 20 nt single-stranded T7 promoter sequence and two repeated 21 nt crRNA sequences.

[0009] Furthermore, sequences complementary to the target RNA strand can be synthesized based on the specific sequence of the target RNA strand to be detected. It is understood that other target RNA strands can also be detected using the system provided by the present invention. The examples of the present invention use the neuronecrosis virus RNA2 as an example, which should not be construed as limiting the scope of protection of the present invention.

[0010] Furthermore, the preparation of the circular probe includes: circularizing a linear PP probe comprising a 20 nt sequence complementary to the target RNA strand, a 20 nt single-stranded T7 promoter sequence, and two repeated 21 nt crRNA sequences by ligase and exonuclease.

[0011] Furthermore, the 5' end of the linear PP probe is phosphorylated.

[0012] Furthermore, the nucleotide sequence of the linear PP probe is shown in SEQ ID NO: 1.

[0013] Furthermore, the method further comprises: annealing and hybridizing the linear PP probe and the splint probe.

[0014] Furthermore, the nucleotide sequence of the splint probe is shown in SEQ ID NO: 2.

[0015] Furthermore, the reaction temperature during detection was 37°C and the reaction time was 2.5 h.

[0016] Furthermore, the nucleotide sequence of the FQ reporter probe is shown in SEQ ID NO: 3.

[0017] This detection system leverages the ability of CRIPSR Cas12a to pre-process pre-crRNA, ingeniously combining rolling circle amplification (RCA) and rolling circle transcription (RCT). The circular probe is designed with a sequence complementary to the target strand, a single-stranded T7 promoter, and DNA sequences corresponding to the crRNA repeats. In the presence of the target strand, Phi29 DNA polymerase uses the CP as a template to amplify the single-stranded T7 promoter into a double-stranded promoter, thereby activating T7 RNA polymerase. Using the phi29 DNA polymerase amplification product as a template, pre-crRNA is transcribed. Cas12a then processes the pre-crRNA into mature crRNA, which then assembles into a RNP complex (Cas12a+crRNA). The RNP uses the phi29 DNA polymerase amplification product, the ssDNA, as an activator, activating its flanking activity. This cleaves the reporter molecule, releasing a fluorescent signal and achieving exponential signal amplification. Furthermore, crRNA production occurs later than the production of the activator ssDNA (single-strand DNA), preventing RNP formation in the early stages of the reaction. This allows for efficient accumulation of amplicons and improves detection sensitivity.

[0018] Compared with the prior art, the present invention has the following beneficial effects: 1. High Sensitivity: In one-pot reactions, amplification and detection often interfere with each other, especially at low concentrations. During the initial amplification phase, Cas proteins cleave amplicons (either forward or sideways), preventing their accumulation and reducing detection sensitivity. However, the generation and accumulation of amplicons in the present invention are prerequisites for crRNA production, effectively overcoming these limitations of one-pot detection and achieving highly sensitive detection.

[0019] 2. Signal re-amplification: The Cas12 tangent product can cyclically activate the circular probe to achieve signal re-amplification.

[0020] 3. High specificity: signals are generated only when complementary targets are recognized.

[0021] 4. Simple and convenient: This invention can realize true one-tube, one-step detection. It only requires sample mixing and constant temperature reaction, without the need for complex instruments and professional operation, and is easy to realize portable and on-site detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 Schematic diagram of the working principle of a CRISPR detection system that integrates target chain amplification and crRNA synthesis into one tube in an embodiment of the present invention; Figure 2 This is the urea-PAGE result of circular probe preparation in a CRISPR detection system that integrates target chain amplification and crRNA synthesis in one tube in an embodiment of the present invention; Figure 3 This is the feasibility analysis result of a CRISPR detection system that integrates target chain amplification and crRNA synthesis into one tube in an embodiment of the present invention; Figure 4 This is the specificity analysis result of a CRISPR detection system that integrates target chain amplification and crRNA synthesis into one tube in an embodiment of the present invention; Figure 5 This is the sensitivity analysis result of a CRISPR detection system that integrates target chain amplification and crRNA synthesis in one tube in an embodiment of the present invention. DETAILED DESCRIPTION

[0024] The technology provided by the present invention is further described in detail and completely below with reference to the accompanying drawings and embodiments.

[0025] The present invention relates to a CRISPR detection system that integrates target strand amplification and crRNA synthesis in a single tube. The system includes a circular probe containing a target strand complementary sequence, a single-stranded T7 promoter, and a double crRNA repeat sequence. It can simultaneously amplify the activator and generate crRNA, achieving exponential signal amplification of the detection signal.

[0026] like Figure 1 As shown, the working principle of the ring probe is as follows: 1. The target strand (using neural necrosis virus RNA2 as an example in the present embodiment) binds to its complementary sequence, activates Phi29 DNA polymerase, and performs rolling circle amplification along the circular probe to generate a double-stranded T7 promoter.

[0027] 2. T7 RNA polymerase recognizes the T7 promoter and transcribes pre-crRNA using the rolling circle amplification product as a template.

[0028] 3. The pre-crRNA is processed into mature crRNA by Cas12a, and the two are assembled into an RNP complex, using the rolling circle amplification product as an activator to activate its side-cutting activity and cut the reporter probe.

[0029] The CRISPR detection system without exogenous synthetic gRNA detection process mainly includes the following steps: 1. Circular probe preparation: Linear PP is prepared into a circular probe through the action of ligase and exonuclease.

[0030] 2. Reaction system configuration: Add target RNA, circular probe, phi29 DNA polymerase, T7 RNA polymerase, Cas12a, NTP mix, rNTP mix, FQ probe, and reaction buffer to a 384-well plate.

[0031] 3. Reaction and signal detection: Set the reaction temperature to 37°C and monitor the fluorescence signal (λ ex =492 nm; λ em =520 nm).

[0032] The embodiments described below are exemplary and are only used to explain the present invention, and are not to be construed as limiting the present invention.

[0033] The experimental methods in the following examples are conventional methods unless otherwise specified. The experiments were usually carried out under conventional conditions. The experimental materials used in the following examples were commercially available unless otherwise specified.

[0034] Example 1: Nucleic acid chain sequence information and preparation All nucleic acid chains were synthesized by Sangon Biotech Co. Ltd., and their sequence information is shown in Table 1. All nucleic acid chains were diluted to 10 μM in nuclease-free water and stored at −20°C.

[0035] Table 1 Oligo sequence information

[0036] Example 2: Preparation of circular probes The PP probe and splint were mixed in a 1× annealing buffer (20 mM NaCl, 10 mM Tris-HCl, pH 7.5) at a molar concentration ratio of 1:1.2. The mixture was denatured at 95°C for 1 min and slowly cooled to room temperature (25°C).

[0037] PP probe circularization: Add 1 μL T4 DNA ligase and 1× ligase reaction buffer to the hybridized chain, react at 25°C for 1 h, then at 85°C for 20 min, and terminate the reaction.

[0038] Removal of linear DNA: Add 1 μL each of exonuclease I and exonuclease III and the corresponding 1× reaction buffer to the ligation product, react at 37°C for 1 h, then at 85°C for 20 min, and terminate the reaction.

[0039] The product was purified by ethanol precipitation as follows: a. Pre-cool anhydrous ethanol, 75% ethanol, and sodium acetate at 4°C; b. Add 1 / 10 volume of NaAc (3 M, pH = 5.2) based on the volume of the DNA sample to balance the negative charge of the DNA; c. Add 3 volumes of pre-cooled anhydrous ethanol, then add 1 μL of glycogen, and gently invert to mix. At this point, white flocs will be visible. Place in a -80°C refrigerator overnight to freeze and precipitate. d. Centrifuge at 13200 rpm for 5 min, observe for white precipitate, and aspirate the supernatant. e. Add 1 mL of ice-cold 75% ethanol, flick off the pellet at the bottom of the tube, centrifuge at 13,200 rpm for 5 minutes, and aspirate the supernatant. Repeat once. f. Seal the centrifuge tube and dry it in a freeze dryer; g. Add an appropriate amount of nuclease-free water to dissolve the precipitate, analyze the results by urea-PAGE, measure the concentration, and store at -20°C.

[0040] Figure 2 Prepare a urea-PAGE gel electrophoresis diagram for the circular probe, where lane 1 is the marker, lanes 2 and 3 are the hybridization products of the PP probe and splint, and lane 4 is the final prepared circular probe.

[0041] Example 3: Feasibility verification of the detection system.

[0042] The complete experimental system is shown in Table 2.

[0043] Set up a positive experimental group and a control group with target RNA, CP, Phi29 DNAP, T7 RNAP, and Cas12a missing components. Use molecular biology-grade nuclease-free water to replace the missing components.

[0044] The reaction mixture was incubated at 37°C for 2.5 h, and the fluorescence signal (λ ex : 492nm; λ em : 520 nm).

[0045] Test results such as Figure 3As shown, there is a very significant difference between the positive experimental group and the other experimental groups. There is no obvious signal increase in the control groups except experimental group 5. Experimental group 5 also has a certain signal increase when the system contains or does not contain T7 RNA polymerase, which is speculated to be due to the certain transcription ability of phi29 DNA polymerase.

[0046] Table 2 Biosensor detection system

[0047] Example 4: Detection system specificity and sensitivity evaluation 1. Select infectious hematopoietic necrosis virus ( Infectious Hematopoietic Necrosis Virus , IHNV), viral hemorrhagic septicemia virus ( Viral haemorrhagic septicemia virus , VHSV) two fish RNA viruses, Klebsiella pneumoniae ( Klebsiella pneumoniae ), Vibrio parahaemolyticus ( Vibrio parahaemolyticus ) and Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) as a control sample for specificity detection.

[0048] 2. The neuronecrosis virus, K.pneumoniae , V. parahaemolyticus , P.aeruginosa The 16sRNA and simulated IHNV and VHSV were used as templates to configure the reaction system as shown in Table 2 to evaluate its specificity.

[0049] 3. Add different concentrations of target to initiate the reaction and evaluate the sensitivity.

[0050] 4. Use molecular biology grade nuclease-free water as a negative control.

[0051] 5. Fluorescence detection (λ ex : 492 nm;λ em : 520 nm), and fluorescence was collected every 2 min.

[0052] Specificity assessment results such as Figure 4 As shown, only the positive experimental group had an obvious signal increase, and no obvious signal was observed in other samples, indicating that the detection system has high specificity.

[0053] The sensitivity evaluation results are as follows Figure 5 As shown in the figure, when the detection target concentration is as low as 10 aM, a detection signal that is clearly different from the negative one can still be observed, indicating that the detection system has high sensitivity detection capability.

[0054] The above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the technical solutions. Those skilled in the art should understand that modifications or equivalent replacements of the technical solutions of the present invention that do not depart from the purpose and scope of the technical solutions of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A CRISPR detection system that integrates target chain amplification and crRNA synthesis into one tube, characterized in that: include: Circular probe; after binding to the target, the circular probe is amplified into a double strand by phi29 DNA polymerase and initiates transcription by T7 RNA polymerase to produce pre-crRNA, which is then processed by Cas12a into mature crRNA, and recognizes the ssDNA amplification product, cuts the FQ reporter probe, and generates a fluorescent signal; The sequence of the circular probe includes a sequence complementary to the target RNA chain, a single-stranded T7 promoter sequence, and a DNA sequence corresponding to the crRNA repeat sequence.

2. The CRISPR detection system integrating target chain amplification and crRNA synthesis into one tube according to claim 1, characterized in that Also includes: The fluorescence signal was monitored by BioTek H1 microplate reader, where λ ex =492 nm; λ em =520 nm.

3. The CRISPR detection system integrating target chain amplification and crRNA synthesis into one tube according to claim 1, characterized in that The circular probe includes a 20 nt sequence complementary to the target RNA, a 20 nt single-stranded T7 promoter sequence and two repeated 21 nt crRNA sequences.

4. The CRISPR detection system integrating target chain amplification and crRNA synthesis into one tube according to claim 3, characterized in that The preparation of the circular probe includes: circularizing a linear PP probe comprising a 20 nt sequence complementary to the target RNA strand, a 20 nt single-stranded T7 promoter sequence and two repeated 21 nt crRNA sequences through the action of a ligase and an exonuclease.

5. The CRISPR detection system integrating target chain amplification and crRNA synthesis into one tube according to claim 4, characterized in that The 5' end of the linear PP probe is phosphorylated.

6. The CRISPR detection system integrating target chain amplification and crRNA synthesis into one tube according to claim 5, characterized in that The nucleotide sequence of the linear PP probe is shown in SEQ ID NO:

1.

7. The CRISPR detection system integrating target chain amplification and crRNA synthesis into one tube according to claim 4, characterized in that Also includes: The linear PP probe anneals and hybridizes with the splint probe.

8. The CRISPR detection system integrating target chain amplification and crRNA synthesis into one tube according to claim 7, characterized in that The nucleotide sequence of the splint probe is shown in SEQ ID NO:

2.

9. The CRISPR detection system integrating target chain amplification and crRNA synthesis in one tube according to claim 1, characterized in that The reaction temperature during detection was 37°C and the reaction time was 2.5 h.

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