A rapid detection method for eccDNA based on CRISPR technology

Abstract

The present invention belongs to the field of biotechnology and relates to a method for rapid detection of eccDNA based on CRISPR technology. The method uses a CRISPR-Cas9 system to target eccDNA containing a target sequence, performs single-stranded DNA probe-assisted enzyme modification at the incision of the target site, directly starts rolling circle amplification of the target eccDNA at a constant temperature, and then uses a CRISPR-Cas14 system to target and bind to the rolling circle amplification product in a single-stranded state, stimulates the trans-cleavage activity of Cas14, and realizes secondary amplification of the signal. Since the present invention has a multi-step specific recognition and signal amplification process in the detection process, it is not necessary to digest the linear DNA present in the genome before detecting the eccDNA, which significantly shortens the detection time of the extrachromosomal circular DNA, reduces the detection cost, can achieve a detection limit of 1fM level, and can successfully detect the eccDNA carrying tumor disease-related gene fragments in tumor cells.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection, and specifically relates to a rapid detection method of eccDNA based on CRISPR technology. Background Art

[0002] Extrachromosomal circular DNA (eccDNA) carrying random genomic fragments is widely present in different cancer types. The amplification of eccDNA promotes tumor genetic heterogeneity and accelerates tumor evolution. With the advancement of analytical technology, eccDNA has recently been shown to be a multifunctional molecule with multiple properties. The unique topological structure and genetic characteristics of eccDNA provide new ideas for cancer monitoring, early diagnosis, treatment and prediction. In existing studies, the main means of analyzing eccDNA is circular DNA sequencing. This method can obtain sequence information of abundant circular DNA in tumor tissues or normal tissues for comparative analysis, thereby revealing tumor pathogenesis and even predicting cancer progression. However, circular DNA sequencing methods also have some limitations, such as complex operation, long sequencing cycle, high price, etc., and cannot perform targeted rapid diagnosis. In recent years, molecular biology technology has achieved rapid development in rapid diagnosis or in vitro diagnosis of diseases. The CRISPR-Cas system has become a powerful tool for gene editing and molecular detection due to its excellent nucleic acid targeting properties. It has the advantages of low cost, simplicity, portability, high sensitivity and high specificity, and is called the "new generation of molecular diagnostic technology."

[0003] Since the CRISPR-Cas system has specific recognition, cis-cleavage and non-specific trans-cleavage capabilities, it has realized the detection of nucleic acid targets such as DNA and RNA, as well as non-nucleic acid targets such as proteins, exosomes, cells and small molecules, and has gradually developed into an independent category of molecular detection. The present invention has developed an in vitro detection method for eccDNA, which utilizes the characteristic that eccDNA itself is circular, combines the specific recognition function of CRISPR-Cas9, amplifies the target circular DNA, and then utilizes the high-fidelity binding characteristics of Cas14a and the trans-cleavage activity after binding to the target site to amplify the signal of the target site, and converts the signal into a detectable fluorescent signal, which is expected to use molecular biological methods to achieve cancer diagnosis and prediction through the detection of eccDNA.

[0004] The existing eccDNA detection and analysis method is mainly circular DNA sequencing (Circle-seq). This research method has obvious limitations: (a) The analysis cycle is long. After obtaining DNA from cells or blood, linear DNA must be digested with nuclease before eccDNA amplification, and the digestion time takes 1-2 weeks. In addition to rolling circle amplification, gene library preparation and data analysis, the sample delivery and detection cycle is as long as 1.5-2 months. (b) The detection cost is high. Due to the need for long-term experimental operation and the need for gene sequencing instruments, eccDNA sequencing analysis is expensive. (c) The detection is weakly targeted. Since the sequencing analysis of eccDNA sequences all circular DNA present in the cell, the detection is less targeted. The occurrence of some diseases is only related to one or several gene fragments existing in eccDNA. The pathological characteristics can be analyzed by detecting this one or several gene fragments. If gene sequencing methods are used to detect such eccDNA every time, a long and high-cost sequencing process is required, and only a small part of the large amount of sequencing data obtained is used, resulting in low resource utilization efficiency. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a rapid detection method for eccDNA based on CRISPR technology.

[0006] In order to achieve the purpose of the present invention, we will adopt the following technical solution to implement it:

[0007] A CRISPR-based eccDNA rapid detection method, without digesting linear DNA in the genome, comprises the following steps:

[0008] S1. Using CRISPR technology, Cas9n-sgRNA is bound to the target site of the target double-stranded circular DNA;

[0009] S2. Synthesize a single-stranded DNA probe with a length of 40 nt according to the target site sequence information, and bind the 40 nt single-stranded DNA probe to the non-complementary strand of the sgRNA;

[0010] S3, using a 40 nt single-stranded DNA probe as a template, Klenow large fragmentase was used to extend the non-complementary strand of sgRNA at the target site;

[0011] S4, starting from the non-complementary chain extension region of sgRNA and using a 20nt single-stranded DNS probe corresponding to the 40nt single-stranded DNA probe as a primer, a strand displacement reaction is performed on the target double-stranded circular DNA to convert the target double-stranded circular DNA into single-stranded circular DNA;

[0012] S5. Add rolling circle amplification primers to the reaction system, and use Ph i29 to perform rolling circle amplification on the single-stranded circular DNA obtained by chain substitution to obtain a rolling circle amplification product;

[0013] S6. Using CRISPR technology, Cas14a-sgRNA and reporter probe are bound to the target site of the rolling circle amplification product to stimulate trans-cleavage activity and cleave the reporter probe;

[0014] S7. Fix the reaction system to a fixed volume and perform fluorescence detection using a fluorescence spectrophotometer.

[0015] As a preferred embodiment of the present invention, the specific operation steps of the eccDNA rapid detection method are as follows:

[0016] S21, select the target site on the target double-stranded circular DNA, and synthesize the sgRNA of Cas9n protein according to the sequence information of the target site;

[0017] S22, take a 1.5mL Eppendorf tube, set the reaction system to 20μL, add Cas9n protein and sgRNA, the final concentration is 50nM, add 10×Cas9n Buffer 2μL, incubate at 37℃ for 20min, so that Cas9n protein and sgRNA can be combined;

[0018] S23, add the target double-stranded circular DNA to the reaction system at a final concentration of 5 nM, and incubate at 37°C for 30 min to allow Cas9n-sgRNA to bind to the target site of the target double-stranded circular DNA;

[0019] S24, add 1 μL of a 40 nt single-stranded DNA probe synthesized according to the target site sequence information to the reaction system, so that the final concentration of the 40 nt single-stranded DNA probe is 500 nM, and incubate at 37°C for 20 min to allow the 40 nt single-stranded DNA probe to bind to the non-complementary strand of the sgRNA;

[0020] S25, add 2 μL dNTP, 1 μL Kl enow large fragment enzyme and corresponding reaction buffer to the reaction system, use 40 nt single-stranded DNA probe as template, and use Kl enow large fragment enzyme to extend the non-complementary chain of sgRNA;

[0021] S26, add 2 μL proteinase K to the reaction system and incubate at 52°C for 60 min to allow Cas9n-sgRNA to fall off from the target site;

[0022] S27, incubate the entire reaction system at 95°C for 10 min to inactivate proteinase K;

[0023] S28, after the reaction system is cooled to room temperature, 1 μL of a 20 nt single-stranded DNA probe corresponding to the 40 nt single-stranded DNA probe is added to make the final concentration of the 20 nt single-stranded DNA probe 500 nM, and then 1 μL of Ph i29 DNA polymerase and the corresponding reaction buffer are added to start rolling circle amplification, and the rolling circle is incubated at 30° C. for 48 hours to obtain a rolling circle amplification product;

[0024] S29, on the rolling circle amplification product, select the target site, and synthesize the sgRNA of Cas14a protein according to the sequence information of the target site;

[0025] S210, take a 1.5mL Eppendorf tube, set the reaction system to 40μL, add Cas14a protein and sgRNA, the final concentration of Cas14a is 50nM, the final concentration of sgRNA is 100nM, add 10×Cas14a Buffer 4μL, incubate at 37°C for 20min, so that Cas14a protein and sgRNA combine;

[0026] S211, add 20 μL of rolling circle amplification product to the reaction system, and then add 2 μL of reporter probe to make the final concentration of the reporter probe 2 μM, incubate at 37 ° C for 30 min, so that Cas14a-sgRNA binds to the target site of the rolling circle amplification product, stimulates trans-cleavage activity, and cuts the reporter probe;

[0027] S212. The reaction system was fixed to 100 μL, and fluorescence detection was performed using a fluorescence spectrophotometer.

[0028] As a preferred embodiment of the present invention, the incubation time in step S211 is adjusted according to the concentration of the rolling circle amplification product to be tested. When the concentration of the rolling circle amplification product to be tested is low, the incubation time is extended to 4 hours.

[0029] As a preferred embodiment of the present invention, the 40 nt single-stranded DNA probe in step S24 is bound to the non-complementary strand of the sgRNA by base complementary pairing.

[0030] As a preferred embodiment of the present invention, the detection sensitivity is improved by increasing the density of binding Cas14a on the rolling circle amplification product.

[0031] As a preferred embodiment of the present invention, the 20 nt single-stranded DNA probe sequence is: 5'-CTAAG GATGC GTGTAATTGC-3'.

[0032] As a preferred embodiment of the present invention, the reporter probe sequence is: FAM-TTTTTTTTTTTT-BHQ1.

[0033] As a preferred embodiment of the present invention, the 40 nt single-stranded DNA probe sequence is: 5'-CTAAG GATGC GTGTAATTGC ATCTC TCTCC TTCTA GCCTC-3'.

[0034] As a preferred embodiment of the present invention, the target double-stranded circular DNA includes but is not limited to plasmid DNA, cell lysate DNA, animal and plant tissue DNA, etc.

[0035] Beneficial Effects

[0036] 1. Since the present invention targets eccDNA containing a specific sequence for amplification, and does not amplify linear DNA and other eccDNA without target sites, it is not necessary to digest linear DNA in advance during detection. In addition, there is no step of constructing a sequencing library and testing on a machine. Therefore, compared with the circular DNA sequencing method, the reaction time will be greatly shortened, the efficiency of the entire reaction system will be improved, and the test cycle can be shortened to less than 60 hours;

[0037] 2. The eccDNA detection method of the present invention can amplify the target eccDNA at a constant temperature, with low dependence on large-scale detection instruments. The main detection cost is in the sgRNA synthesis step of Cas14a and Cas9n proteins. Compared with the circular DNA sequencing method, the detection cost is greatly reduced;

[0038] 3. The present invention realizes accurate amplification and detection of target sites through two targeted combinations of Cas9n and Cas14a, overcomes the influence of off-target effect of Cas9n protein, and can perform targeted detection of eccDNA containing specific target sequences;

[0039] 4. The present invention undergoes two-step signal amplification processes of rolling circle amplification and Cas14a trans-cleavage, and can improve the detection sensitivity by increasing the density of Cas14a bound to the RCA product, and the detection limit of double-stranded circular DNA can reach 1fM. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Schematic diagram of the recognition site of Cas9n-sgRNA on eccDNA;

[0041] Figure 2 Schematic diagram of the rolling circle amplification process mediated by single-stranded DNA probe;

[0042] Figure 3 Schematic diagram of Cas14a binding to the rolling circle amplification product, stimulating trans-cleavage activity, and cleaving the reporter probe to generate a fluorescent signal;

[0043] Figure 4is the detection limit achieved by this system for pUC19 circular double-stranded DNA;

[0044] Figure 5 It is the detection limit that the present invention can achieve for Ph iX174 circular double-stranded DNA under the influence of interfering DNA; Figure 6 This is the detection result of the present invention for three types of eccDNA containing sites related to human hypopharyngeal squamous cell carcinoma. The experimental group of eccDNA fragments containing three gene sites showed different degrees of signal enhancement compared with the control group, indicating that the detection of disease-related eccDNA has been achieved. DETAILED DESCRIPTION

[0045] The present invention will be further described below in conjunction with specific embodiments and drawings.

[0046] As an embodiment of the present invention, Figures 1 to 4 As shown, a double-stranded circular DNA (pUC19 plasmid) with a length of 2.7 kb was subjected to rolling circle amplification using a single-stranded DNA probe-mediated method.

[0047] Step 1: According to the sequence information of the 2.7kb double-stranded circular DNA (pUC19 plasmid), after selecting the target site, synthesize the sgRNA of the Cas9n protein, obtain the sgRNA primer of the Cas9n protein corresponding to the target site from Sangon Biotech Co., Ltd., and use the sgRNA primer to synthesize and transcribe the sgRNA of the Cas9n protein of the target site.

[0048] Cas9n-sg-F: 5'-TAATA CGACT CACTA TAGGG ACAGA ATCAG GGGAT AACGC GTTTTAGAGC TAGAA ATAGC AAGTT AAA-3';

[0049] Cas9n-sg-R: 5'-GCACC GACTC GGTGC CACTT TTTCA AGTTG ATAAC GGACT AGCCTTATTT TAACT TGCTA TTTCT AGC-3';

[0050] Cas14a-sg-F: 5'-GAAAT TAATA CGACT CACTA TAGGG TTCAC TGATA AAGTG GAGAACCGCT TCACC AAAAG CTGTC CCTTA GGGGA TTAGA ACTTG AGTGA AGGTG GGCTG CTTGC ATCAGCCTAA-3';

[0051] Cas14a-sg-R:5'-TCACG CTCGT CGTTT GGTAT GTTGC ATTCC TTCAT TCTTT CAAATGAATT TGTTT CGAGG GTTAC TTTCC GAAGA AAGCA CTTCT CGACA TTAGG CTGAT GCAAG CAGCCCACCT-3';

[0052] Step 2: Take a 1.5mL Eppendorf tube, set the reaction system to 20μL, add Cas9n protein and sgRNA corresponding to the pUC19 double-stranded circular DNA target site, the final concentration is 50nM, add 10×Cas9n Buffer 2μL, incubate at 37℃ for 20min to allow Cas9n protein to combine with sgRNA.

[0053] Step 3: Add pUC19 double-stranded circular DNA to the reaction system at a final concentration of 5 nM and incubate at 37°C for 30 min to allow Cas9n-sgRNA to bind to the target site of the target double-stranded circular DNA.

[0054] Step 4: Add 1 μL of a 40-nt single-stranded DNA probe synthesized according to the target site sequence to the reaction system, so that the final concentration of the 40-nt single-stranded DNA probe is 500 nM. Incubate at 37°C for 20 minutes to allow the 40-nt single-stranded DNA probe to bind to the non-complementary chain of the sgRNA.

[0055] 40 nt single-stranded DNA probe sequence: 5'-CTAAG GATGC GTGTA ATTGC GCGTT ATCCC CTGATTCTGT-3'.

[0056] Step 5: Add 2 μL dNTP, 1 μL Klenow large fragmentase and corresponding reaction buffer to the reaction system, and use the 40nt single-stranded DNA probe as a template to extend the sgRNA non-complementary chain through Klenow large fragmentase.

[0057] Step 6: Add 2 μL of proteinase K to the reaction system and incubate at 52°C for 60 min to allow Cas9n-sgRNA to fall off from the target site.

[0058] Step 7: Incubate the entire reaction system at 95°C for 5 min to inactivate proteinase K.

[0059] Step 8: After the reaction system is cooled to room temperature, add 1 μL of 20 nt single-stranded DNA probe (complementary to the black chain in the figure) corresponding to the 40 nt single-stranded DNA probe, so that the final concentration of the 20 nt single-stranded DNA probe is 500 nM, then add 1 μL Ph i29 DNA polymerase and the corresponding reaction buffer and 1 μL rolling circle amplification primer, so that the final concentration of the rolling circle amplification primer is 500 nM, start chain replacement and rolling circle amplification, and roll at 30°C for 48 hours;

[0060] 20 nt single-stranded DNA probe sequence: 5'-CTAAG GATGC GTGTA ATTGC-3'.

[0061] Rolling circle amplification primer sequence: GTAAA GCCTG GGGTG CCTAA TG;

[0062] Step 9: Take a 1.5mL Eppendorf tube, set the reaction system to 40μL, add Cas14a protein and sgRNA corresponding to Ph iX174 double-stranded circular DNA, the final concentration of Cas14a is 50nM, the final concentration of sgRNA is 100nM, add 10×Cas14a Buffer 4μL, incubate at 37°C for 20min, so that Cas14a protein and sgRNA combine;

[0063] Step 10: Add 20 μL of the rolling circle amplification product obtained in step 7 to the reaction system, and then add 2 μL of the reporter probe (FQ Reporter) to make the final concentration of the reporter probe 2 μM, and incubate at 37 ° C for 30 min to 4 h to allow Cas14a-sgRNA to bind to the target site on the rolling circle amplification product, stimulate trans-cleavage activity, and cut the reporter probe;

[0064] FQ reporter sequence: FAM-TTTTTTTTTTTT-BHQ1;

[0065] Step 11: The reaction system was fixed to 100 μL, and fluorescence detection was performed using a fluorescence spectrophotometer, wherein the fluorescence spectrophotometer was set to an excitation wavelength of 492 nm, a PMT voltage of 650 V, and a scan speed of 240 nm / min.

[0066] As an embodiment of the present invention, Figures 1 to 3 as well as Figure 5 As shown, the circular double-stranded DNA of Ph iX174 with a length of 5.4 kb was used as the detection object, and different concentrations of Ph iX174 were detected.

[0067] Step 1: After selecting the target site according to the sequence information of the double-stranded circular DNA of Ph iX174, the sgRNA primers of the Cas9n protein and Cas14a protein corresponding to the target site were synthesized from Sangon Biotech Co., Ltd., and the sgRNA of the Cas9n protein and Cas14a protein of the target site were synthesized and transcribed using the sgRNA primers;

[0068] Cas9n-sg-F: 5'-TAATA CGACT CACTA TAGGG AAGGT CATGC GGCAT ACGCT GTTTTAGAGC TAGAA ATAGC AAGTT AAA-3';

[0069] Cas9n-sg-R: GCACC GACTC GGTGC CACTT TTTCA AGTTG ATAAC GGACT AGCCTTATTT TAACT TGCTA TTTCT AGC-3';

[0070] Cas14a-sg-F: 5'-GAAAT TAATA CGACT CACTA TAGGG TTCAC TGATA AAGTG GAGAACCGCT TCACC AAAAG CTGTC CCTTA GGGGA TTAGA ACTTG AGTGA AG-GTG GGCTG CTTGCATCAG CCTAA-3';

[0071] Cas14a-sg-R: 5'-AACAA TTTAG ACATG GCGCC GTTGC ATTCC TTCAT TCTTT CAAATGAATT TGTTT CGAGG GTTAC TTTCC GAAGA AAGCA CTTCT CGACA TTAGG CTGAT GCAAG CAGCCCACCT-3';

[0072] Step 2: Take a 1.5mL Eppendorf tube, set the reaction system to 20μL, add Cas9n protein and sgRNA corresponding to the double-stranded circular DNA target site of Ph iX174, the final concentration is 50nM, add 10×Cas9n Buffer 2μL, incubate at 37℃ for 20min, so that Cas9n protein and sgRNA can be combined;

[0073] Step 3: Add Ph iX174 double-stranded circular DNA to the reaction system with a final concentration of 1pM-1fM gradient concentration. At the same time, add 10nM concentration of M13 double-stranded circular DNA and 1pM linear Ph iX174 double-stranded DNA to the reaction system, and incubate at 37°C for 30min to allow Cas9n-sgRNA to bind to the target site of the target double-stranded circular DNA.

[0074] Step 4: Add 1 μL of a 40-nt single-stranded DNA probe synthesized according to the target site sequence to the reaction system, so that the final concentration of the 40-nt single-stranded DNA probe is 500 nM, and incubate at 37°C for 20 min to allow the 40-nt single-stranded DNA probe to bind to the non-complementary strand of the sgRNA;

[0075] 40 nt single-stranded DNA probe sequence: 5′-CTAAG GATGC GTGTA ATTGC AGCGT ATGCC GCATGACCTT-3′;

[0076] Step 5: Add 2 μL dNTP, 1 μL Kl enow large fragmentase and corresponding reaction buffer to the reaction system, and use the 40 nt single-stranded DNA probe as a template to extend the sgRNA non-complementary chain through Kl enow large fragmentase;

[0077] Step 6: Add 2 μL of proteinase K to the reaction system and incubate at 52°C for 60 min to allow Cas9n-sgRNA to fall off from the target site;

[0078] Step 7: Incubate the entire reaction system at 95°C for 5 min to inactivate proteinase K;

[0079] Step 8: After the reaction system is cooled to room temperature, add 1 μL of a 20 nt single-stranded DNA probe (complementary to the black chain in the figure) corresponding to the 40 nt single-stranded DNA probe, so that the final concentration of the 20 nt single-stranded DNA probe is 500 nM, then add 1 μL Ph i29 DNA polymerase and the corresponding reaction buffer and 1 μL rolling circle amplification primer, so that the final concentration of the rolling circle amplification primer is 500 nM, start chain replacement and rolling circle amplification, and roll at 30°C for 48 hours;

[0080] 20 nt single-stranded DNA probe sequence: 5′-CTAAG GATGC GTGTA ATTGC-3′;

[0081] Rolling circle amplification primer sequence: CAAAA CGGCA GAAGC CTGAA TG;

[0082] Step 9: Take a 1.5mL Eppendorf tube, set the reaction system to 40μL, add Cas14a protein and sgRNA corresponding to Ph iX174 double-stranded circular DNA, the final concentration of Cas14a is 50nM, the final concentration of sgRNA is 100nM, add 10×Cas14a Buffer 4μL, incubate at 37°C for 20min, so that Cas14a protein and sgRNA combine;

[0083] Step 10: Add 20 μL of the rolling circle amplification product obtained in step 7 to the reaction system, and then add 2 μL of the reporter probe (FQ Reporter) to make the final concentration of the reporter probe 2 μM, incubate at 37 ° C for 30 min, so that Cas14a-sgRNA binds to the target site on the rolling circle amplification product, stimulates trans-cleavage activity, and cuts the reporter probe;

[0084] FQ reporter sequence: FAM-TTTTTTTTTTTT-BHQ1;

[0085] Step 11: The reaction system was fixed to 100 μL, and fluorescence detection was performed using a fluorescence spectrophotometer, wherein the fluorescence spectrophotometer was set to an excitation wavelength of 492 nm, a PMT voltage of 650 V, and a scan speed of 240 nm / min.

[0086] As an embodiment of the present invention, Figures 1 to 3 as well as Figure 6 As shown, human hypopharyngeal squamous cell carcinoma cells (FaDu) were used as the detection object to detect eccDNA associated with hypopharyngeal squamous cell carcinoma (HSCC);

[0087] Step 1: Based on the sequencing information of eccDNA of FaDu cells, three target gene fragments (TP53, CDCA3, TMEM184B) related to HSCC disease and present on eccDNA were screened. According to the sequence information of the gene fragment to be tested on eccDNA, sgRNA primers of Cas9n protein and Cas14a protein corresponding to the target site on eccDNA were synthesized from Sangon Biotech Co., Ltd., and sgRNA of Cas9n protein at the target site was synthesized and transcribed using sgRNA primers;

[0088] TP53-Cas9n-sg-F: 5'-TAATA CGACT CACTA TAGGG GCGCC ATTGC ACTCT AGCCTGTTTT AGAGC TAGAA ATAGC AAGTT AAA-3';

[0089] <h2 style=";text-align:left;direction:ltr">CDCA3-Cas9n-sg-F:5'-TAATA CGACT CACTA TAGGG AGAGC CTGTT CTGCC CCCAGGTTTT AGAGC TAGAA ATAGC AAGTT AAA-3';<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0090] <h2 style=";text-align:left;direction:ltr"> TMEM184B-Cas9n-sg-F:5'-TAATA CGACT CACTA TAGGG TAGTG ATAGG CAAAGGCCCA GTTTT AGAGC TAGAA ATAGC AAGTT AAA-3';<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0091] <h2 style=";text-align:left;direction:ltr"> Cas9n-sg-R:GCACC GACTC GGTGC CACTT TTTCA AGTTG ATAAC GGACT AGCCTTATTT TAACT TGCTA TTTCT AGC-3';<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0092] <h2 style=";text-align:left;direction:ltr"> Cas14a-sg-F:5'-GAAAT TAATA CGACT CACTA TAGGG TTCAC TGATA AAGTG GAGAACCGCT TCACC AAAAG CTGTC CCTTA GGGGA TTAGA ACTTG AGTGA AG-GTG GGCTG CTTGCATCAG CCTAA-3';<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0093] <h2 style=";text-align:left;direction:ltr"> TP53-Cas14a-sg-R:5'-TAAAG CAGGA GGATA ACTTG GTTGC ATTCC TTCAT TCTTTCAAAT GAATT TGTTT CGAGG GTTAC TTTCC GAAGA AAGCA CTTCT CGACA TTAGG CTGAT GCAAGCAGCC CACCT-3';<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0094] <h2 style=";text-align:left;direction:ltr"> CDCA3-Cas14a-sg-R:5'-CAGGG ATACT GAGGA ATGGC GTTGC ATTCC TTCAT TCTTTCAAAT GAATT TGTTT CGAGG GTTAC TTTCC GAAGA AAGCA CTTCT CGACA TTAGG CTGAT GCAAGCAGCC CACCT-3';<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0095] TMEM184B-Cas14a-sg-R: 5'-GACTT TGCAG AAGTG ACTGA GTTGC ATTCC TTCATTCTTT CAAAT GAATT TGTTT CGAGG GTTAC TTTCC GAAGA AAGCA CTTCT CGACA TTAGG CTGATGCAAG CAGCC CACCT-3';

[0096] Step 2: Take a 1.5mL Eppendorf tube, set the reaction system to 30μL, add Cas9n protein and sgRNA of Cas9n protein corresponding to three eccDNA target sites to different components, the final concentration is 50nM, add 10×Cas9n Buffer 3μL, incubate at 37℃ for 20min:, so that Cas9n protein and sgRNA can be combined;

[0097] Step 3: Add 10 μg of genomic DNA extracted from FaDu cells to the reaction system, which contains all the eccDNA in the cells, and incubate at 37°C for 30 minutes to allow Cas9n-sgRNA to bind to the target site of the target eccDNA;

[0098] Step 4: Add 1 μL of a 40 nt single-stranded DNA probe synthesized according to the target site sequence to the reaction system to make the final concentration of the probe 500 nM, and incubate at 37°C for 20 min to allow the probe to bind to the non-complementary strand of the sgRNA;

[0099] TP53 locus 40 nt single-stranded DNA probe sequence: 5′-CTAAG GATGC GTGTA ATTGC AGGCT AGAGTGCAAT GGCGC-3′;

[0100] The sequence of the 40 nt single-stranded DNA probe at CDCA3 locus was: 5′-CTAAG GATGC GTGTA ATTGC CTGGG GGCGAAACAG GCTCT-3′;

[0101] TMEM184B locus 40 nt single-stranded DNA probe sequence: 5′-CTAAG GATGC GTGTA ATTGC TGGGCCTTTG CCTAT CACTA-3′;

[0102] Step 5: Add 2 μL dNTP, 1 μL Klenow large fragment enzyme and corresponding reaction buffer to the reaction system, and use the 40 nt probe as a template to extend the sgRNA non-complementary chain through Klenow large fragment enzyme;

[0103] Step 6: Add 2 μL of proteinase K to the reaction system and incubate at 52°C for 60 min to allow Cas9n-sgRNA to fall off from the target site;

[0104] Step 7: Incubate the entire reaction system at 95°C for 5 min to inactivate proteinase K;

[0105] Step 8: After the reaction system is cooled to room temperature, add 1 μL of a 20 nt single-stranded DNA probe (complementary to the black chain in the figure) corresponding to the 40 nt single-stranded DNA probe, so that the final concentration of the 20 nt single-stranded DNA probe is 500 nM, then add 1 μL Ph i29 DNA polymerase and the corresponding reaction buffer and 1 μL rolling circle amplification primer, so that the final concentration of the rolling circle amplification primer is 500 nM to start chain replacement and rolling circle amplification, and roll at 30°C for 48 hours;

[0106] 20 nt single-stranded DNA probe sequence: 5′-CTAAG GATGC GTGTA ATTGC-3′;

[0107] TP53 locus rolling circle amplification primer sequence: AATCG CTTGA ACCCA GGAGG CA

[0108] CDCA3 site rolling circle amplification primer sequence: AAGCG GCCTT CACCC CTAAG TG

[0109] Primer sequence for rolling circle amplification of TMEM184B locus: CTCTG TCCTT CCCCA GACAA GG

[0110] Step 9: Take a 1.5mL Eppendorf tube, set the reaction system to 50μL, add Cas14a protein and sgRNA of Cas14a protein corresponding to three eccDNA target sites respectively, the final concentration of Cas14a is 50nM, the final concentration of sgRNA is 100nM, add 10×Cas14a Buffer 5μL, incubate at 37°C for 20min, so that Cas14a protein and sgRNA can be combined;

[0111] Step 10: Add 30 μL of the rolling circle amplification product obtained in step 7 to the reaction system, and then add 2 μL of the reporter probe (FQ Reporter) to make the final concentration of the reporter probe 2 μM, incubate at 37 ° C for 30 min, so that Cas14a-sgRNA binds to the target site on the rolling circle amplification product, stimulates trans-cleavage activity, and cuts the reporter probe;

[0112] FQ reporter sequence: FAM-TTTTTTTTTTTT-BHQ1;

[0113] Step 11: The reaction system was fixed to 100 μL, and fluorescence detection was performed using a fluorescence spectrophotometer, wherein the fluorescence spectrophotometer was set to an excitation wavelength of 492 nm, a PMT voltage of 650 V, and a scan speed of 240 nm / min.

[0114] The technical solution of the present invention is described in detail above in conjunction with the embodiments / drawings, but the present invention is not limited to the above technical solution. For ordinary technicians in this technical field, after knowing the contents recorded in the present invention, they can make several equivalent changes and substitutions without departing from the principle of the present invention. These equivalent changes and substitutions should also be regarded as belonging to the protection scope of the present invention.

Claims

1. A rapid detection method for eccDNA based on CRISPR technology, characterized in that: The detection method is used for eccDNA detection for non-diagnostic purposes, and includes the following steps without digesting linear DNA in the genome: S1. Using CRISPR technology, Cas9n-sgRNA is bound to the target site of the target double-stranded circular DNA; S2. Synthesize a single-stranded DNA probe with a length of 40 nt according to the target site sequence information, and bind the 40 nt single-stranded DNA probe to the non-complementary strand of the sgRNA; S3, using a 40 nt single-stranded DNA probe as a template, Klenow large fragment enzyme was used to extend the non-complementary strand of sgRNA at the target site; S4, starting from the non-complementary chain extension region of sgRNA and using a 20nt single-stranded DNA probe corresponding to the 40nt single-stranded DNA probe as a primer, a strand displacement reaction is performed on the target double-stranded circular DNA to convert the target double-stranded circular DNA into single-stranded circular DNA; S5, adding a rolling circle amplification primer to the reaction system, using Phi29 to perform rolling circle amplification on the single-stranded circular DNA obtained by strand substitution, to obtain a rolling circle amplification product; S6. Using CRISPR technology, Cas14a-sgRNA and reporter probe are bound to the target site of the rolling circle amplification product to stimulate trans-cleavage activity and cleave the reporter probe; S7. Fix the reaction system to a fixed volume and perform fluorescence detection using a fluorescence spectrophotometer.

2. According to claim 1, a CRISPR-based eccDNA rapid detection method is characterized in that: The specific operation steps of the eccDNA rapid detection method are as follows: S21, select the target site on the target double-stranded circular DNA, and synthesize the sgRNA of Cas9n protein according to the sequence information of the target site; S22, take a 1.5mL Eppendorf tube, set the reaction system to 20μL, add Cas9n protein and sgRNA, the final concentration is 50nM, add 10×Cas9n Buffer 2μL, incubate at 37℃ for 20min, so that Cas9n protein and sgRNA can be combined; S23, add the target double-stranded circular DNA to the reaction system at a final concentration of 5 nM, and incubate at 37°C for 30 min to allow Cas9n-sgRNA to bind to the target site of the target double-stranded circular DNA; S24, add 1 μL of a 40 nt single-stranded DNA probe synthesized according to the target site sequence information to the reaction system to make the final concentration of the single-stranded DNA probe 500 nM, and incubate at 37°C for 20 min to allow the single-stranded DNA probe to bind to the non-complementary strand of the sgRNA; S25, add 2 μL dNTP, 1 μL Klenow large fragment enzyme and corresponding reaction buffer to the reaction system, use 40 nt single-stranded DNA probe as template, and use Klenow large fragment enzyme to extend the non-complementary chain of sgRNA; S26, add 2 μL proteinase K to the reaction system and incubate at 52°C for 60 min to allow Cas9n-sgRNA to fall off from the target site; S27, incubate the entire reaction system at 95°C for 10 min to inactivate proteinase K; S28, after the reaction system is cooled to room temperature, 1 μL of the 20nt probe corresponding to the 40nt single-stranded DNA probe is added to make the final concentration of the 20nt probe 500 nM, and then 1 μL of Phi29 DNA polymerase and the corresponding reaction buffer are added to start rolling circle amplification, and the rolling circle is incubated at 30°C for 48 hours to obtain the rolling circle amplification product; S29, on the rolling circle amplification product, select the target site, and synthesize the sgRNA of Cas14a protein according to the sequence information of the target site; S210, take a 1.5mL Eppendorf tube, set the reaction system to 40μL, add Cas14a protein and sgRNA, the final concentration of Cas14a is 50nM, the final concentration of sgRNA is 100nM, add 10×Cas14a Buffer 4μL, incubate at 37°C for 20min, so that Cas14a protein and sgRNA combine; S211, add 20 μL of rolling circle amplification product to the reaction system, and then add 2 μL of reporter probe to make the final concentration of the reporter probe 2 μM, incubate at 37 ° C for 30 min, so that Cas14a-sgRNA binds to the target site of the rolling circle amplification product, stimulates trans-cleavage activity, and cuts the reporter probe; S212. The reaction system was fixed to 100 μL, and fluorescence detection was performed using a fluorescence spectrophotometer.

3. The method for rapid detection of eccDNA based on CRISPR technology according to claim 2, characterized in that: The incubation time in step S211 is adjusted according to the concentration of the rolling circle amplification product to be tested. When the concentration of the rolling circle amplification product to be tested is low, the incubation time is extended to 4 hours.

4. The method for rapid detection of eccDNA based on CRISPR technology according to claim 2, characterized in that: The single-stranded DNA probe in step S24 is bound to the non-complementary strand of the sgRNA by base complementary pairing.

5. A CRISPR-based eccDNA rapid detection method according to claim 1 or 2, characterized in that: The detection sensitivity is improved by increasing the density of Cas14a bound to the rolling circle amplification product.

6. The method for rapid detection of eccDNA based on CRISPR technology according to claim 1, characterized in that: The 20 nt single-stranded DNA probe sequence is: 5'-CTAAG GATGC GTGTA ATTGC-3'.

7. The method for rapid detection of eccDNA based on CRISPR technology according to claim 1, characterized in that: The reporter probe sequence is: FAM-TTTTTTTTTTTT-BHQ1.

8. The method for rapid detection of eccDNA based on CRISPR technology according to claim 1, characterized in that: The target double-stranded circular DNA includes plasmid DNA, cell lysate DNA, animal tissue DNA or plant tissue DNA.

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