An electrochemical method for detecting DNA damage based on CRISPR-Cas12a system

Abstract

The application discloses an electrochemical method for detecting DNA damage based on a CRISPR-Cas12a system. Normal DNA can activate the activity of the CRISPR-Cas12a complex, resulting in the cutting of the DNA probe fixed on the electrode surface. The cut DNA probe cannot undergo HCR reaction in the presence of hairpin DNA H1 and H2, thereby generating a smaller electrochemical signal of methylene blue; damaged DNA cannot completely activate the activity of the CRISPR-Cas12a complex, at this time, the uncut DNA probe induces HCR in the presence of hairpin DNA H1 and H2 and generates longer double-stranded DNA, a large number of methylene blue molecules are combined with the double-stranded DNA, thereby generating a stronger electrochemical signal. The damage degree of DNA corresponds to the light damage level thereof, the electrochemical signal of methylene blue and the light damage level of DNA are in a linear relationship in the range of 0.021-0.42 kJ m ‑2 , and the lowest detection limit is 0.0088 kJ m ‑2 .

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biosensing, and particularly relates to an electrochemical method for detecting DNA damage based on a CRISPR-Cas12a system. BACKGROUND

[0002] The integrity of DNA is crucial for maintaining genetic stability and cell function. DNA damage is ubiquitous in living organisms and can lead to genetic mutations and even cancer and other diseases. Ultraviolet radiation can cause damage to DNA in cells. When DNA absorbs ultraviolet radiation, cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) are formed between adjacent pyrimidine bases. The formation of the above photoproducts interferes with the normal functions of cells, such as DNA replication and transcription processes, so detecting the process of ultraviolet radiation-induced DNA damage has important biological significance.

[0003] At present, there are various methods for detecting ultraviolet-induced DNA damage, such as chromatography-mass spectrometry, surface plasmon resonance, electrochemical impedance spectroscopy, and fluorescence method. Among them, chromatography-mass spectrometry has been used to quantitatively analyze CPDs and 6-4PPs in genomic DNA. By inserting a fluorescent probe into DNA, fluorescence detection of DNA photodamage levels can be achieved.

[0004] The CRISPR-Cas (clustered regularly interspaced short palindromic repeats and associated proteins) system has become a powerful tool for gene editing in recent years and has been widely used in the field of biosensing. Among them, CRISPR-Cas12a is an RNA-guided endonuclease that can specifically recognize target DNA and activate transcleavage activity. At present, most researches are usually focused on the recognition and quantification of specific DNA sequences by the CRISPR-Cas12a system. There is no report on the use of the CRISPR-Cas12a system to detect DNA damage. SUMMARY

[0005] The purpose of the present application is to provide an electrochemical method for detecting DNA damage based on the CRISPR-Cas12a system. The DNA probe is fixed on the surface of the electrode. After being cut by the CRISPR-Cas12a complex, the hairpin DNA H1 and H2 are added to undergo a hybrid chain reaction (HCR) reaction. The HCR product binds with methylene blue. The strength of the electrochemical signal of methylene blue corresponds to the difference in the activation degree of the CRISPR-Cas12a complex. According to the principle that different damage degrees of DNA correspond to different activation degrees of the CRISPR-Cas12a complex, different DNA photodamage levels are obtained, so it has good application value.

[0006] The application provides an electrochemical method for detecting DNA damage based on a CRISPR-Cas12a system, comprising the following steps:

[0007] S1. Obtain a DNA nucleotide sequence to be detected, construct sample DNA, irradiate a plurality of sample DNAs under an ultraviolet lamp for a preset time to obtain a group of sample DNAs with different damage degrees, mix Cas12a and crRNA to incubate to generate a Cas12a / crRNA complex, then add the sample DNAs with different damage degrees to the complex respectively and incubate to obtain a group of CRISPR-Cas12a complexes with different activation degrees;

[0008] S2. After polishing a gold electrode, drop a mixture of thiolated DNA probes and TCEP (tris (2-carboxyethyl) phosphine) on the electrode surface and incubate, then clean the electrode surface with water and ethanol respectively and blow dry, drop 6-mercapto-1-hexanol and incubate, then clean the electrode surface with water and ethanol respectively and blow dry, drop the CRISPR-Cas12a complexes with different activation degrees obtained in step S1 respectively and incubate to obtain a group of pretreated electrodes;

[0009] S3. Drop a mixture of hairpin DNAs H1 and H2 on the group of pretreated electrodes obtained in step S2 to perform HCR reaction, then clean the electrodes and blow dry, drop methylene blue solution and incubate to obtain a group of methylene blue treated electrodes;

[0010] S4. Test the electrochemical signals of methylene blue in a phosphate buffer (10 mM PBS, pH 7.4) by differential pulse voltammetry in a three-electrode system to obtain the relationship between the light damage level of DNA and the electrochemical signals of methylene blue;

[0011] S5. According to the relationship between the light damage level of DNA and the electrochemical signals of methylene blue obtained in step S4, process the DNA to be detected to obtain the light damage level of the DNA to be detected.

[0012] Further, in step S1, the sample DNA is irradiated under ultraviolet light for a preset time gradient, the time gradient ranges from 0 to 120 min, the ultraviolet wavelength ranges from 230 to 270 nm, and the ultraviolet irradiation intensity ranges from 0.3 to 0.4 W m -2 .

[0013] The process conditions for mixing Cas12a and crRNA to incubate to generate a Cas12a / crRNA complex are as follows: the concentration ratio of Cas12a to crRNA is 1: (0.8-1.2), the incubation time is 20-40 min, and the incubation temperature is 35-40℃;

[0014] The incubation conditions for the Cas12a / crRNA complex and damaged DNA were as follows: the concentration ratio of Cas12a / crRNA complex to damaged DNA was 1:(0.8-1.2), the incubation time was 20-40 min, and the incubation temperature was 35-40℃.

[0015] Further, in step S2, the concentration of the thiolated DNA probe in the mixture of the thiolated DNA probe and TCEP is 0.1–1 μM, and the concentration of TCEP is 0.8–1.2 mM;

[0016] The process conditions for incubating the thiolized DNA probe and TCEP mixture on a gold electrode were as follows: the volume of the thiolized DNA probe and TCEP mixture added was 8–15 μL, the incubation temperature was 20–25 °C, and the incubation time was 8–12 h.

[0017] The process conditions for incubation by adding 6-mercapto-1-hexanol dropwise are as follows: the concentration of 6-mercapto-1-hexanol added dropwise is 0.5-1 mM, the volume is 30-50 μL, the incubation temperature is 20-25℃, and the incubation time is 0.5-1 h.

[0018] The incubation conditions for CRISPR-Cas12a complexes with different activation levels were as follows: the volume of CRISPR-Cas12a complexes with different activation levels added was 8–15 μL, the incubation temperature was 35–40 °C, and the incubation time was 100–150 min.

[0019] Further, in step S3, the concentration of hairpin DNA H1 used in the mixture of hairpin DNA H1 and H2 is 1.5–2.5 μM, the concentration of hairpin DNA H2 is 1.5–2.5 μM, and the concentration ratio during mixing is 1:(0.8–1.2); the amount of hairpin DNA H1 and H2 mixture added is 8–15 μL, the HCR reaction temperature is 35–40 °C, and the time is 100–150 min.

[0020] The concentration of methylene blue solution added was 15–25 mM, the amount added was 20–30 μL, the incubation temperature was 20–25 °C, and the incubation time was 20–40 min.

[0021] Furthermore, in step S4, a three-electrode system is used, wherein the working electrode is a methylene blue treated electrode, the reference electrode is an Ag / AgCl reference electrode, and the counter electrode is a platinum wire electrode; the electrolyte solution is 10mM PBS with pH 7.4.

[0022] Further, step S5 includes the following steps:

[0023] A. Prepare the Cas12a / crRNA complex, then add the DNA to be tested and incubate to obtain the activated CRISPR-Cas12a complex;

[0024] The process conditions for preparing the Cas12a / crRNA complex are the same as those for the incubation of Cas12a and crRNA in step S1 to generate the Cas12a / crRNA complex.

[0025] The incubation conditions are the same as those in step S1 when the Cas12a / crRNA complex is mixed with the damaged DNA for incubation.

[0026] B. Polish the gold electrode, add a mixture of thiolized DNA probe and TCEP (tris(2-carboxyethyl)phosphine), incubate overnight, wash the electrode surface with water and ethanol respectively and dry it, then add 6-mercapto-1-hexanol for incubation; wash the electrode surface with water and ethanol again and dry it, then add activated CRISPR-Cas12a complex for incubation to obtain the complex pretreated electrode.

[0027] The process conditions for this step are the same as those for the corresponding step in step S2.

[0028] C. Add DNAH1 and H2 to the electrode pretreated with the complex and carry out the HCR reaction. After the reaction is complete, wash the electrode and dry it. Add methylene blue solution and incubate to obtain the methylene blue treated electrode.

[0029] The process conditions for this step are the same as those for the corresponding step in step S3.

[0030] D. Based on the relationship between the photodamage level of DNA and the electrochemical signal of methylene blue, the photodamage level of the DNA to be tested is obtained by using a three-electrode system in PBS to test the electrochemical signal of methylene blue using differential pulse voltammetry; the process conditions in this step are the same as those in the corresponding step in step S4.

[0031] The principle of this invention:

[0032] Ultraviolet (UV) radiation can induce the formation of cyclobutanepyrimidine dimers (CPDs) and pyrimidine (6-4)pyrimidinone photoproducts (6-4PPs) between adjacent pyrimidine bases in DNA. When the sample DNA is undamaged, the CRISPR-Cas12a system can be activated normally; in this case, the thiolized DNA probes immobilized on the electrode surface are cleaved, preventing the HCR reaction from being initiated in the presence of hairpin DNA H1 and H2, resulting in a small electrochemical signal upon the introduction of methylene blue. When UV irradiation damages the sample DNA, the CRISPR-Cas12a system cannot be activated and cannot properly cleave the surface-immobilized DNA probes. In this case, the DNA probes initiate the HCR reaction in the presence of H1 and H2, first opening the stem-loop structure of hairpin H1, and then the stem-loop structure of hairpin H2. This generates a large amount of double-stranded DNA on the electrode surface; the generated double-stranded DNA interacts with methylene blue molecules through electrostatic interactions or insertion mechanisms, producing a significant electrochemical signal. The degree of DNA damage can be measured by the level of photodamage. As the degree of damage changes, the electrochemical signal of methylene blue changes, and the difference in the electrochemical signal of methylene blue corresponds to different levels of DNA photodamage.

[0033] The beneficial effects of this invention are:

[0034] (1) This invention is the first to use the CRISPR-Cas12a system to detect DNA damage, and different degrees of damage correspond to different levels of photodamage;

[0035] (2) The HCR reaction initiated by the electrode surface in the method of the present invention is highly dependent on the integrity of the DNA probe, which reduces background noise and improves analytical performance;

[0036] (3) The method for detecting DNA photodamage level based on the CRISPR-Cas12a system has the advantages of high sensitivity and wide linear range, and is expected to be used for the detection of DNA damage in cells. Attached Figure Description

[0037] Figure 1 This is a schematic diagram illustrating the electrochemical detection principle of DNA damage based on the CRISPR-Cas12a system in the embodiments.

[0038] Figure 2 The following is a gel electrophoresis image from the example, where lane 1: DNA probe without thiol group, lane 2: H1, lane 3: H2, lane 4: DNA probe + hairpin H1, lane 5: hairpin H1 + H2, lane 6: DNA probe + hairpin H1 + H2, lane 7: CRISPR-Cas12a complex + DNA probe + H1 + H2, lane 8: damaged T 21 +Cas12a / crRNA+DNA probe+H1+H2;

[0039] Figure 3 A represents the damage T caused by different ultraviolet radiation doses in the example. 21 The corresponding electrochemical signal at that time; Figure 3 B represents the linear relationship curve between the electrochemical signal and the ultraviolet dose. Detailed Implementation

[0040] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0041] The embodiments of the present invention employ DNAT 21 Using DNA samples as test samples, an electrochemical method for assessing DNA damage was constructed based on the enzymatic cleavage reaction of DNA probes by the CRISPR-Cas12a complex. The principle is as follows: Figure 1 As shown. First, thiolized DNA probes are immobilized on the surface of a gold electrode. When the DNA probe to be tested... 21 When damaged, the CRISPR-Cas12a complex cannot be properly activated, preventing the DNA probe from being cleaved. Intact DNA probes undergo an HCR reaction in the presence of hairpin DNA H1 and H2, generating longer double-stranded DNA. Subsequently, methylene blue molecules intercalate into the double-stranded DNA, producing a strong electrochemical signal. Normal or undamaged DNA can activate the enzymatic cleavage activity of the CRISPR-Cas12a complex, causing the surface-fixed DNA probe to be cleaved. In the presence of hairpin DNA H1 and H2, the HCR reaction cannot be triggered, resulting in a smaller amount of methylene blue binding to the electrode surface, thus producing a weaker electrochemical signal.

[0042] Among them, DNA T 21 The nucleotide sequences of crRNA, thiolized DNA probes, and hairpin DNA H1 and H2 are shown in the table below.

[0043] Table 1 Nucleotide Sequences

[0044]

[0045] Example 1: Validation method for detecting DNA damage based on the CRISPR-Cas12a system

[0046] Take 10 groups of 0.1 mL, 0.5 μM T 21 The intensity exposed to 254 nm is 0.35 W / m. -2 Ten groups of UV damage T values ​​were obtained by applying UV light for 0 min, 1 min, 5 min, 7.5 min, 10 min, 15 min, 20 min, 30 min, 60 min, and 120 min. 21 ;

[0047] 150 nM crRNA and 150 nM Cas12a were mixed and incubated at a concentration ratio of 1:1 for 30 min at a temperature of 37 °C to obtain the Cas12a / crRNA complex.

[0048] T 10 groups of UV-damaged T 21 Cas12a / crRNA complexes were added to the DNA, with the concentration ratio of Cas12a / crRNA complex to damaged DNA being 1:1. The incubation time was 30 min and the incubation temperature was 37℃, resulting in 10 groups of CRISPR-Cas12a complexes with different activation levels.

[0049] Gold electrodes were polished on a polishing cloth using 0.3 μm and 0.05 μm alumina slurries, and then ultrasonically cleaned in water and ethanol. A 0.5 μM thiolated DNA probe was mixed with a 1 mM TCEP solution and incubated in the dark for 1 h. Then, 10 μL of the thiolated DNA probe and TCEP mixture was added dropwise to the polished gold electrode and incubated overnight at room temperature. The gold electrode was rinsed with PBS, and 10 μL of 1.0 mM 6-mercapto-1-hexanol was added and incubated for 0.5 h. Ten groups of CRISPR-Cas12a complexes with different activation levels were added dropwise to the electrode surface and incubated at 37 °C for 120 min. Then, a hairpin DNA H1 and H2 mixture was added to the electrode and reacted at 37 °C for 2 h. Finally, the electrode was immersed in a 20 mM methylene blue solution and incubated at room temperature for 30 min. Finally, differential pulse voltammetry was performed in 10 mM PBS within the range of -0.5 V to 0 V to obtain the relationship between the degree of DNA damage and the electrochemical signal of methylene blue.

[0050] Damage T at different UV doses was detected 21 The corresponding electrochemical signal is as follows: Figure 3 As shown in Figure A, the linear relationship curve between the electrochemical signal and the ultraviolet dose is as follows: Figure 3 B. Because in this embodiment, the degree of DNA damage can be represented using ultraviolet (UV) dose, therefore... Figure 3 As shown in A and 3B, the oxidation current of methylene blue increases with increasing UV dose, ranging from 0.021 to 0.42 kJ / m². -2 Within the specified range, the oxidation current exhibits a linear relationship with the ultraviolet dose, with the linear equation being i(μA) = 7.27UV dose(kJ / m²). -2 )+1.64(R 2 =0.9959). Based on S / N=3, the lowest detection limit of the method is 0.0088 kJ / m³. -2 .

[0051] Example 2: Gel electrophoresis experiment to verify the occurrence of HCR reaction

[0052] Eight experimental groups were set up, each with eight lanes for gel electrophoresis;

[0053] The first experimental group consisted of DNA probes;

[0054] The second experimental group was hairpin DNA H1;

[0055] The third experimental group was hairpin DNA H2;

[0056] The fourth experimental group consisted of DNA probes and hairpin DNA H1.

[0057] The fifth experimental group consisted of hairpin DNA H1 and hairpin DNA H2.

[0058] The sixth experimental group consisted of DNA probe + hairpin DNA H1 + hairpin DNA H2;

[0059] The 7th experimental group consisted of CRISPR-Cas12a complex + DNA probe + hairpin DNA H1 + hairpin DNA H2;

[0060] The 8th experimental group was the damaged T 21 +Cas12a / crRNA+DNA probe+hairpin DNA H1+hairpin DNA H2;

[0061] Gel electrophoresis results as follows Figure 2 As shown, the single bands in lanes 1, 2, and 3 correspond to the DNA probe, H1, and H2, respectively. The appearance of a new, slower-migrating band in lane 4 indicates that the DNA probe successfully opened the H1 card. The brighter bands in lane 5 correspond to H1 and H2, while the slow-migrating, blurry bands indicate a weak HCR reaction. The continuous bright bands with slow migration rates in lane 6 correspond to the occurrence of the HCR reaction. Lane 7 is similar to lane 5, with normal T... 21 Successful activation of the CRISPR-Cas12a complex enzyme activity led to DNA probe cleavage, resulting in a weak HCR reaction. Lane 8 is similar to lane 6, damaging T... 21 The inability to fully bind to the Cas12a-crRNA complex results in the DNA probe being almost uncut, thereby inducing the HCR reaction.

Claims

1. An electrochemical method for detecting DNA damage based on a CRISPR-Cas12a system, characterized in that, Includes the following steps: S1. Obtain the nucleotide sequence of the DNA to be tested and construct sample DNA; irradiate several sample DNAs under a UV lamp for a preset time to obtain a set of sample DNAs with different degrees of damage; mix Cas12a with crRNA and incubate to generate Cas12a / crRNA complex, then add sample DNAs with different degrees of damage to it and incubate to obtain a set of CRISPR-Cas12a complexes with different activation levels. S2. After polishing the gold electrode, a mixture of thiolized DNA probe and TCEP (tris(2-carboxyethyl)phosphine) was added to the electrode surface and incubated. The electrode surface was cleaned with water and ethanol respectively and dried. Then, 6-mercapto-1-hexanol was added and incubated. The electrode surface was cleaned with water and ethanol again and dried. Then, CRISPR-Cas12a complexes with different activation levels obtained in step S1 were added and incubated to obtain a set of pretreated electrodes. S3. Add a mixture of hairpin DNA H1 and H2 to a set of pretreated electrodes obtained in step S2, perform HCR reaction, clean the electrodes and dry them after the reaction is complete, add methylene blue solution and incubate to obtain a set of methylene blue treated electrodes; S4. The set of methylene blue treatment electrodes obtained in step S3 were tested in PBS using differential pulse voltammetry with a three-electrode system to obtain the relationship between the photodamage level of DNA and the electrochemical signal of methylene blue. S5. Based on the relationship between the photodamage level of DNA obtained in step S4 and the electrochemical signal of methylene blue, the DNA to be tested is processed to obtain the photodamage level of the DNA to be tested. Step S5 includes the following steps: A. Prepare the Cas12a / crRNA complex, then add the DNA to be tested and incubate to obtain the activated CRISPR-Cas12a complex; B. Polish the gold electrode, add a mixture of thiolized DNA probe and TCEP (tris(2-carboxyethyl)phosphine), incubate overnight, wash the electrode surface with water and ethanol respectively and dry it, then add 6-mercapto-1-hexanol for incubation; wash the electrode surface with water and ethanol again and dry it, then add the activated CRISPR-Cas12a complex obtained in step A for incubation to obtain the pretreated electrode. C. Add hairpin DNA H1 and H2 to the pretreated electrode obtained in step B, perform HCR reaction, clean the electrode and dry it after the reaction is complete, add methylene blue solution and incubate to obtain methylene blue treated electrode; D. Based on the relationship between the photodamage level of DNA and the electrochemical signal of methylene blue obtained in step S4, the methylene blue treatment electrode obtained in step C was tested in PBS using differential pulse voltammetry and a three-electrode system to obtain the photodamage level of the DNA to be tested. The source of the light damage is ultraviolet radiation.

2. The electrochemical method for detecting DNA damage based on the CRISPR-Cas12a system according to claim 1, characterized in that, In step S1, the sample DNA is irradiated under a preset time gradient under a UV lamp. The time gradient range is 0–120 min, the UV wavelength range is 230–270 nm, and the UV irradiation intensity range is 0.3–0.4 W / m². −2 .

3. The electrochemical method for detecting DNA damage based on the CRISPR-Cas12a system according to claim 1, characterized in that, In step S1, the process conditions for generating the Cas12a / crRNA complex by mixing and incubating Cas12a and crRNA are as follows: the concentration ratio of Cas12a to crRNA is 1:(0.8~1.2), the incubation time is 20~40 min, and the incubation temperature is 35~40 ℃. The incubation conditions for the Cas12a / crRNA complex and damaged DNA were as follows: the concentration ratio of Cas12a / crRNA complex to damaged DNA was 1:(0.8~1.2), the incubation time was 20~40 min, and the incubation temperature was 35~40 ℃.

4. The electrochemical method for detecting DNA damage based on the CRISPR-Cas12a system according to claim 1, characterized in that, In step S2, the concentration of the thiolated DNA probe in the TCEP mixture is 0.1~1 μM and the concentration of TCEP is 0.8~1.2 mM. The process conditions for adding the thiolated DNA probe and TCEP mixture to the gold electrode and incubating are as follows: the volume of the thiolated DNA probe and TCEP mixture added is 8~15 μL, the incubation temperature is 20~25 ℃, and the incubation time is 8~12 h.

5. The electrochemical method for detecting DNA damage based on the CRISPR-Cas12a system according to claim 1, characterized in that, In step S2, the process conditions for incubation by adding 6-mercapto-1-hexanol are as follows: the concentration of 6-mercapto-1-hexanol added is 0.5~1 mM, the volume is 30~50 μL, the incubation temperature is 20~25 ℃, and the incubation time is 0.5~1 h.

6. The electrochemical method for detecting DNA damage based on the CRISPR-Cas12a system according to claim 1, characterized in that, In step S2, the process conditions for incubating CRISPR-Cas12a complexes with different activation levels are as follows: the volume of CRISPR-Cas12a complexes with different activation levels added is 8~15 μL, the incubation temperature is 35~40 ℃, and the incubation time is 100~150 min.

7. The electrochemical method for detecting DNA damage based on the CRISPR-Cas12a system according to claim 1, characterized in that, In step S3, the concentration of hairpin DNA H1 used in the mixture of hairpin DNA H1 and H2 is 1.5~2.5 μM, and the concentration ratio of hairpin DNA H2 is 1:(0.8~1.2). The amount of hairpin DNA H1 and H2 mixture added is 8~15 μL, the HCR reaction temperature is 35~40 ℃, and the time is 100~150 min.

8. The electrochemical method for detecting DNA damage based on the CRISPR-Cas12a system according to claim 1, characterized in that, In step S3, the concentration of methylene blue solution added is 15~25 mM, the amount added is 20~30 μL, the incubation temperature is 20~25℃, and the incubation time is 20~40 min.

9. The electrochemical method for detecting DNA damage based on the CRISPR-Cas12a system according to claim 1, characterized in that, In step S4, a three-electrode system is used, wherein the working electrode is a methylene blue treated electrode, the reference electrode is an Ag / AgCl reference electrode, and the counter electrode is a platinum wire electrode; the electrolyte solution is 10 mM PBS with pH 7.4.

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