阅读说明：本技术 一种基于CRISPR-Cas9点特异性切割可视化检测ssRNA或ssDNA的方法 (Method for visually detecting ssRNA or ssDNA based on CRISPR-Cas9 point-specific cleavage ) 是由 侯长军 霍丹群 汪显峰 陈晓龙 杨眉 罗小刚 于 2020-05-22 设计创作，主要内容包括：本发明公开了一种基于CRISPR-Cas9点特异性切割可视化检测ssRNA或ssDNA的方法,该方法通过CRISPR/Cas9的可编程切口功能实现ssRNA和ssDNA的点特异性切割,以产生所需要的单链DNA片段,该片段在3’端具有特定的序列,可以用作引物来激发EXPAR进行指数扩增,从而实现对不同靶标序列ssRNA和ssDNA可视化检测。本发明具有成本低,操作简便,效率高,灵敏度高和特异性强,结果读取简单,减少了对热循环仪器和精密信号采集仪器的依赖。为单链核苷酸的检测提供了新思路和新选择,也将在临床诊断和生物医学研究中发挥关键作用,具有良好的应用前景。(The invention discloses a method for visually detecting ssRNA or ssDNA based on CRISPR-Cas9 point-specific cleavage, which realizes the point-specific cleavage of ssRNA and ssDNA through the programmable nicking function of CRISPR/Cas9 to generate a required single-stranded DNA fragment, wherein the fragment has a specific sequence at the 3' end and can be used as a primer to excite EXPAR to carry out exponential amplification, thereby realizing the visual detection of ssRNA and ssDNA with different target sequences. The invention has the advantages of low cost, simple operation, high efficiency, high sensitivity, strong specificity and simple result reading, and reduces the dependence on a thermal cycler and a precise signal acquisition instrument. Provides a new idea and a new choice for the detection of single-stranded nucleotide, plays a key role in clinical diagnosis and biomedical research, and has good application prospect.)

1. A method for visually detecting ssRNA or ssDNA based on CRISPR-Cas9 point-specific cleavage, which is characterized by comprising the following steps:

1) mixing target genes to be detected with different concentrations, PAMmer, Cas9 and sgRNA in a buffer solution according to a proper proportion, and incubating for 30-60 min at 37 ℃ to obtain a point-specific cleavage product; the target gene is ssRNA or ssDNA, the PAMmer is matched with a target gene to be detected, the sgRNA comprises an anchoring sequence and a guide region sequence, the anchoring sequence is specifically recognized by Cas9 protein, and the guide region sequence is matched with a fragment of the target gene;

2) adding the point-specific cleavage product obtained in the step 1) into an EXPAR reaction system to perform exponential amplification reaction, and performing phi29DNA polymerase extension and Nb.BbvCI selective endonuclease cleavage to obtain an amplification product rich in a G sequence;

3) adding K-containing sequence to the amplification product of the G-rich sequence obtained in step 2)⁺The buffer and hemin solution were allowed to react thoroughly, and excess ABTS and H were added₂O₂Incubating for 5min, and detecting the absorption intensity of ultraviolet and visible light of the reaction system by using an ultraviolet and visible spectrophotometer;

4) and calculating a regression equation by making a standard curve of the target gene concentration-the absorption intensity of the ultraviolet visible light according to standard solutions of target genes with different concentrations, and calculating the concentration of the target genes in the solution to be detected according to the absorption intensity of the ultraviolet visible light of the solution to be detected.

2. The method for visually detecting ssRNA or ssDNA based on CRISPR-Cas9 point-specific cleavage according to claim 1, wherein the sgRNA is obtained by overlapping and extending two partially complementary DNA single strands to obtain a transcription template, and then transcribing the transcription template.

3. The method for visually detecting ssRNA or ssDNA based on CRISPR-Cas9 point-specific cleavage according to claim 2, wherein the program of overlap extension: multiplying at 95 ℃ for 3 min; 30 thermal cycles: 95 ℃ X20 s, 52 ℃ X30 s, 72 ℃ X25 s; 72 ℃ for 10 min.

4. The method for visually detecting ssRNA or ssDNA based on CRISPR-Cas9 point-specific cleavage according to claim 1, wherein the molar ratio of the PAMmer, Cas9 and sgRNA in step 1) is 1:1: 1.

5. The method for visually detecting ssRNA or ssDNA based on CRISPR-Cas9 point-specific cleavage according to claim 1, wherein the final concentration of the target gene to be detected in step 1) in the reaction system is 100 aM-2.5 nM.

6. The method for visually detecting ssRNA or ssDNA based on CRISPR-Cas9 spot-specific cleavage according to claim 1, wherein the EXPAR reaction system in step 2) comprises 0.3U/. mu.L phi29DNA polymerase, 0.4U/. mu.L Nb.BbvCI, 1 XDNA polymerase buffer, 1 XCutSmart buffer, 0.5mM dNTP and 10nM EXPAR template; the amplification reaction temperature is 30-45 ℃, and the reaction time is 1-2 h; the point-specific cleavage product is used as a primer to match with an EXPAR template, and is extended to synthesize double-stranded DNA.

7. The method for visually detecting ssRNA or ssDNA based on CRISPR-Cas9 spot-specific cleavage according to claim 6, wherein the phi29DNA polymerase can also be Klenow Fragment (exo-) DNA polymerase or vent (exo-) DNA polymerase.

8. The method for visually detecting ssRNA or ssDNA based on CRISPR-Cas9 point-specific cleavage according to claim 1, wherein the reaction temperature in step 3) is 30-45 ℃ and the reaction time is 10-40 min.

9. The method for visually detecting ssRNA or ssDNA based on CRISPR-Cas9 point-specific cleavage according to claim 1, wherein the absorption intensity in step 3) is an ultraviolet absorption signal at 420nm, and the detection range is 400 nm-460 nm.

10. Use of the method according to any of claims 1 to 9 for detecting single base mutations in ssRNA or ssDNA.

Technical Field

The invention relates to the technical field of biomedical monitoring, in particular to a method for visually detecting ssRNA or ssDNA based on CRISPR-Cas9 point-specific cleavage.

Background

For most RNA viruses, the organism is infected in the form of single-stranded RNA (ssrna), and some DNA viruses replicate in the form of single-stranded DNA (ssdna). In addition, many RNAs exist in single stranded form in the human body to perform their specific functions. Therefore, rapid and accurate detection of single-strand specific nucleic acids plays a crucial role in disease diagnosis and pathology analysis. Meanwhile, single base change in the nucleic acid sequence is the genetic basis for drug resistance of bacteria, viruses and tumors and has close relation with various diseases. Therefore, the method can quickly and accurately detect the single base change in the nucleic acid sequence and also has important effects on biological research and disease diagnosis.

Currently, researchers regarding methods for detecting nucleic acids have developed various nucleic acid signal amplification techniques based on nucleic acid polymerases, nucleic acid hydrolases, deoxyribozymes, nanomaterials, and the like. For example, the most common method at present is a signal amplification technique based on Polymerase Chain Reaction (PCR), i.e., adding a molecular beacon into a PCR system and releasing fluorescence by combining the molecular beacon with a PCR product, which not only requires a precise temperature-variable control instrument but also has certain limitations on the detection of single-stranded oligonucleotides. Researchers have also developed a single-cycle nucleic acid signal amplification technology based on ribonuclease H (RNaseH), that is, a molecular beacon with an RNA sequence in its loop is hybridized with a target DNA, and the RNA of the molecular beacon is partially hydrolyzed under the action of RNaseH, thereby releasing a fluorescent group. Although the target sequence of this technique may be a single-stranded oligonucleotide, since it is a single-cycle linear signal amplification technique, the signal amplification efficiency is low and the signal intensity generated within a certain period of time is insufficient, resulting in low detection sensitivity. Thereby limiting the practical application of the above method.

Isothermal amplification techniques developed in recent years are the most commonly used methods for signal amplification in nucleic acid detection, and are simpler and more convenient than PCR techniques in terms of both practical operation and instrument requirements, and they do not depend on sophisticated equipment, and show promising application prospects in clinical and in-situ rapid diagnosis, including Rolling Circle Amplification (RCA), catalytic hairpin assembly technique (CHA), Hybrid Chain Reaction (HCR), Strand Displacement Amplification (SDA) and EXPAR. Among them, the RCA method involves a cumbersome DNA probe ligation process and adjustment of primer-template ratio, the amplification efficiency of CHA and HCR driven by entropy change is low, and background interference is easily caused. The high amplification efficiency of EXPAR and SDA makes them effective nucleic acid detection amplification strategies. However, ssRNA and ssDNA have long nucleotide sequences and undefined 3' ends, which make ssRNA and ssDNA not directly usable as primers for priming an amplification reaction. Therefore, establishing accurate and efficient strategies for measuring ssRNA and ssDNA remains a highly challenging task.

The CRISPR/Cas system is an immune system currently found in most and all archaea to protect against the invasion of foreign substances. CRISPR (clustered regularly interspaced short palindromic repeats) is a clustered regularly interspaced short palindromic repeat, and the system specifically recognizes invading viral nucleic acid and cuts exogenous nucleic acid by using CRISPR-associated protein (Cas protein) to achieve the purpose of defense. In recent years, the CRISPR/Cas system is widely used in studies such as gene editing, gene expression regulation, and gene detection as a very important tool in genetic engineering.

Disclosure of Invention

Aiming at the defects of the existing detection technology, the invention aims to provide a method for visually detecting ssRNA or ssDNA by point-specific cleavage based on CRISPR-Cas9, and aims to solve the problems that the traditional long-chain ssRNA or ssDNA detection is high in cost, dependent on thermal cycling equipment, requires deeper professional knowledge of detection personnel and the like, and ssRNA and ssDNA have longer nucleotide sequences and uncertain 3' ends and cannot be directly applied to isothermal amplification technologies such as Strand Displacement Amplification (SDA), EXPAR and the like, so that the field visual detection of ssRNA and ssDNA becomes possible.

In order to solve the technical problems, the invention adopts the following technical scheme: a method for visually detecting ssRNA or ssDNA based on CRISPR-Cas9 point-specific cleavage, comprising the steps of:

1) mixing target genes to be detected with different concentrations, PAMmer, Cas9 and sgRNA in a buffer solution according to a proper proportion, and incubating for 30-60 min at 37 ℃ to obtain a point-specific cleavage product; the target gene is ssRNA or ssDNA, the PAMmer is matched with a target gene to be detected, the sgRNA comprises an anchoring sequence and a guide region sequence, the anchoring sequence is specifically recognized by Cas9 protein, and the guide region sequence is matched with a fragment of the target gene;

2) adding the point-specific cleavage product obtained in the step 1) into an EXPAR reaction system for amplification reaction, and performing phi29DNA polymerase extension and Nb.BbvCI selective endonuclease cleavage to obtain an amplification product rich in a G sequence;

3) adding K-containing sequence to the amplification product of the G-rich sequence obtained in step 2)⁺The buffer and hemin solution were allowed to react thoroughly, and excess ABTS and H were added₂O₂Incubating for 5min to combine the amplification product rich in the G sequence with hemin to form a G-quadruplex which can fully react, and detecting the absorption intensity of ultraviolet and visible light of the reaction system by using an ultraviolet and visible spectrophotometer;

4) and calculating a regression equation by making a standard curve of the target gene concentration-the absorption intensity of the ultraviolet visible light according to standard solutions of target genes with different concentrations, and calculating the concentration of the target genes in the solution to be detected according to the absorption intensity of the ultraviolet visible light of the solution to be detected.

The invention realizes the point-specific cutting of ssRNA and ssDNA through the programmable nicking function of CRISPR/Cas9 to generate a required single-stranded DNA fragment, the fragment has a specific sequence at the 3' end and can be used as a primer to excite EXPAR for detection, and a spacer region (20nt) complementary with the target ssRNA or ssDNA in the sgRNA can be artificially designed in the system so as to realize the visual detection of the ssRNA and the ssDNA of different target sequences.

Preferably, the sgRNA is obtained by overlapping and extending two partially complementary DNA single strands to obtain a transcription template, and transcribing the transcription template.

Preferably, the program for overlap extension: multiplying at 95 ℃ for 3 min; 30 thermal cycles: 95 ℃ X20 s, 52 ℃ X30 s, 72 ℃ X25 s; 72 ℃ for 10 min.

Preferably, the molar ratio of PAMmer, Cas9 to sgRNA in step 1) is 1:1: 1.

Preferably, the final concentration of the target gene to be detected in step 1) in the reaction system is 100aM to 2.5 nM.

Preferably, the EXPAR reaction system in step 2) comprises 0.3U/. mu.Lphi 29DNA polymerase, 0.4U/. mu.LNb. BbvCI, 1 XDNA polymerase buffer, 1 XCutSmart buffer, 0.5mM dNTP and 10 nMUEXPAR template; the amplification reaction temperature is 30-45 ℃, and the reaction time is 1-2 h; the point-specific cleavage product is used as a primer to match with an EXPAR template, and is extended to synthesize double-stranded DNA. DNA polymerase has both strand extension and displacement functions, and nb.

Preferably, the phi29DNA polymerase is Klenow fragment (exo-) DNA polymerase or vent (exo-) DNA polymerase.

Preferably, the reaction temperature in the step 3) is 30-45 ℃, and the reaction time is 10-40 min.

Preferably, the absorption intensity in step 3) is an ultraviolet absorption signal at 420nm, and the detection range is 400 nm-460 nm.

The invention also provides the application of the method in detecting single base mutation of ssRNA or ssDNA.

The detection principle of the invention is as follows: as shown in fig. 1A, Cas9 assembles with the sgRNA to form a Cas9/sgRNA binary complex. The target ssRNA (or ssDNA) hybridizes to the PAMmer sequence. Subsequently, Cas9 recognizes the "NGG" (PAM) site on the PAMmer sequence and facilitates hybridization of the 20nt guide region sequence of the sgRNA to the ssRNA (or ssDNA) target sequence, forming a 20bp heteroduplex. Cas9 cleaves a specific site near the PAM of the heteroduplex, releasing a point-specific cleavage fragment of the target ssRNA or ssDNA (P1).

As shown in FIG. 1B, using G4-EXPAR (including X-Y-Y region) as a template and a site-specific cleavage fragment P1 (capable of specifically binding to X region) as a primer, under the action of phi29DNA polymerase, the template is extended from the 3 'end to the 5' end, a double-stranded DNA is formed during the extension, and under the action of Nb.BbvCI endonuclease, secondary polymerase extension is activated, and the generated strand displacement effect releases the early P2 and P3 products (cycle 1 in FIG. 1B). In addition, P2 and P3 can also be used as primers, assembled with the Y region in the middle of the G4-EXPAR templateTogether, and through polymerase extension and strand displacement cycles, a large number of amplification products, P2 and P3 products, accumulate (cycle 2 in fig. 1B). Amplification products of G-rich sequences (P2 and P3) at K⁺With the aid of (3), the compound can be combined with hemin to form a G-quadruplex and has the catalytic activity of hydrogen peroxide mimic enzyme. G-quadruplex/hemin catalysis H₂O₂The decomposition produces large amounts of Reactive Oxygen Species (ROS), resulting in the oxidation of ABTS to ABTS^·+The color changes from colorless to bright green. Thus, the Cas-G4EX system can enable label-free, point-specific visual detection of ssRNA and ssDNA.

Compared with the prior art, the invention has the following beneficial effects:

1. the invention realizes the point-specific cutting of ssRNA and ssDNA by the programmable nicking function of CRISPR/Cas 9. So as to generate a required single-stranded DNA fragment, wherein the fragment has a specific sequence at the 3' end and can be used as a primer to excite EXPAR to carry out exponential amplification, thereby realizing the visual detection of ssRNA and ssDNA with any sequence. Therefore, the difficult problems that long-chain ssRNA and ssDNA have no fixed 3' end and cannot be used as primers for carrying out isothermal amplification such as Strand Displacement Amplification (SDA), EXPAR and the like are solved.

2. The detection method integrates the point specificity recognition and cutting of CRISPR/Cas9 and the high amplification efficiency of G4-EXPAR, provides a multi-stage signal amplification strategy, realizes a novel method for detecting ssRNA and ssDNA with high sensitivity, and has the detection limit of 250aM for ssRNA and 100aM for ssDNA. Meanwhile, the Cas-G4EX system has extremely important practical value in actual sample detection. The invention provides a new idea and a new choice for the detection of single-stranded nucleotide, plays a key role in clinical diagnosis and biomedical research, and has good application prospect.

3. The detection system has the advantages of high response speed, high accuracy, good repeatability, simple and convenient operation, high sensitivity, strong specificity and simple result reading, and reduces the dependence on a thermal cycler and a precise signal acquisition instrument.

4. The invention firstly utilizes the single-stranded nucleic acid cleavage activity of the PAMmer assisted CRISPR/Cas9 system for the detection of single base mutation of ssRNA or ssDNA, enlarges the detection and application range of the CRISPR/Cas9 system and provides a theoretical basis of feasibility.

Drawings

FIG. 1 is a schematic diagram of the detection system of the present invention for detecting ssRNA/ssDNA.

FIG. 2 is a graph showing the sensitivity of the detection system of the present invention to ssDNA; FIG. A is a graph of the UV-VIS absorption spectra of ssDNA at different concentrations; panel B is the corresponding standard plot taken for a logarithmic fit.

FIG. 3 is a diagram showing the specific analysis of ssDNA single base mutation by the detection system of the present invention; figure a is a schematic diagram of CRISPR/Cas9 point-specific cleavage of ssDNA; FIG. B is a graph of the UV-visible absorption spectra of different single base mutant ssDNAs; panel C is a graph of uv-vis spectral response of different single base mutant ssDNA.

FIG. 4 is a graph showing the sensitivity of the detection system of the present invention to ssRNA; FIG. A is a graph of the UV-VIS absorption spectra of ssRNA at different concentrations; panel B is the corresponding standard plot taken for a logarithmic fit.

FIG. 5 is a diagram showing the specificity analysis of the single base mutation of ssRNA by the detection system of the present invention; figure a is a schematic diagram of CRISPR/Cas9 point-specific cleavage of ssRNA; FIG. B is a graph of the UV-visible absorption spectra of different single base mutant ssRNAs; FIG. C is a graph of UV-VIS spectral response of different single base mutant ssRNAs.

Detailed Description

The present invention will be described in further detail with reference to examples.

Examples below all nucleic acid sequences were synthesized by Shanghai biological Co., Ltd and purified by high performance liquid chromatography. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, reagents and starting materials for use in the present invention are commercially available.