Method for double-ring detection of lung cancer L858R mutation based on CRISPR / Cas9 and DNA

Abstract

The invention relates to the technical field of biological detection, and discloses a method for double-ring detection of lung cancer L858R mutation based on CRISPR / Cas9 and DNA, and the method comprises the following steps: enriching circulating tumor cells CTC from a blood sample; extracting genomic DNA (deoxyribonucleic acid) of the enriched CTC; carrying out amplification on the DNA fragment containing the L858R mutation by adopting recombinase polymerase amplification RPA (recombinase polymerase amplification); the amplified DNA fragment is hybridized with a recognition ring in a DNA double-ring structure, a DNA double ring is composed of the recognition ring and a report ring, and a part of region of the recognition ring is hybridized with a corresponding fragment of the report ring to form a staggered double-chain region; when L858R mutation exists, the mutated DNA and a recognition ring are hybridized to activate a CRISPR / Cas9 system, and under the guidance of sgRNA, Cas9 cuts hybrid double strands and releases a report ring; carrying out rolling circle amplification RCA under the action of DNA polymerase by taking the released report ring as a template; a fluorescent probe is used for detecting an RCA amplification product, and whether L858R mutation exists or not is judged according to a fluorescence signal. The CRISPR / Cas9 system is combined with a DNA double-ring structure, so that the ultra-sensitive and specific detection on the lung cancer L858R mutation is realized.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological detection, and particularly relates to a method for detecting lung cancer L858R mutation based on CRISPR / Cas9 and DNA double-loop. BACKGROUND

[0002] Lung cancer is a malignant tumor with high morbidity and mortality worldwide, and non-small cell lung cancer (NSCLC) is the main type. EGFR L858R mutation is a common single nucleotide polymorphism (SNP) in NSCLC, and accurate detection thereof is of great significance for the development of individualized treatment plan, prognosis evaluation and early diagnosis of lung cancer patients. However, the existing detection techniques have many limitations when facing the low-abundance L858R mutation in CTCs.

[0003] The commonly used SNP detection methods, such as NGS technology, are the gold standard for clinical identification of DNA mutations, but the detection process is complicated, time-consuming, requires professional bioinformatics analysis personnel and expensive sequencing equipment, and the cost is high. The qPCR has strict requirements for experimental conditions, is easily affected by primer design, amplification efficiency and other factors, and thus the accuracy and repeatability of the detection results are poor. The method based on nucleic acid probe hybridization has limited sensitivity and is difficult to detect low-abundance mutations. When detecting SNP in CTCs, the number of CTCs in the peripheral blood of patients is extremely small, only a few to tens of CTCs per milliliter of blood, and the amount of genomic DNA extracted from such a small amount of CTCs is extremely low, only about 6 picograms per cell, which makes the conventional amplification and detection method prone to false negative results, seriously affecting the early diagnosis of lung cancer and the monitoring of treatment effect.

[0004] The CRISPR / Cas9 system has great potential in gene editing and nucleic acid detection due to its strong recognition ability and precise cleavage activity. It can specifically recognize and cleave double-stranded DNA (dsDNA) under the guidance of single guide RNA (sgRNA). However, Cas9 itself lacks trans-cleavage activity and cannot non-specifically cleave other substrate molecules after recognizing the target sequence, which limits its application in nucleic acid signal amplification and multiple target detection in complex biological samples. At the same time, the development of DNA nanostructure-based biosensors has brought new ideas for nucleic acid detection, but current research on structural reconstruction (allosteric) mainly focuses on the application of DNA nanostructures, and the research on combining CRISPR / Cas9 system with DNA nanostructures (such as DNA double loop) for lung cancer L858R mutation detection is still in the exploratory stage. In the prior art, although there have been attempts to combine CRISPR-Cas system with nucleic acid isothermal amplification technology, there are still many challenges in precisely combining CRISPR / Cas9 with DNA double loop with specific topological structure and achieving efficient detection, such as how to ensure the stability of DNA double loop structure when working with CRISPR / Cas9 system, how to optimize the reaction conditions of their combination to achieve high-sensitivity detection of low-abundance L858R mutation, etc. These problems have not been effectively solved. SUMMARY

[0005] The present application aims to provide a method for detecting lung cancer L858R mutation based on CRISPR / Cas9 and DNA double loop, to solve the problems of time-consuming, high cost, need for professional personnel and high-precision instruments in existing single nucleotide polymorphism (SNP) detection methods such as next-generation sequencing (NGS) technology, quantitative real-time polymerase chain reaction (qPCR) and nucleic acid probe hybridization-based methods; and when detecting SNP in circulating tumor cells (CTCs), due to the extremely small number of CTCs in the peripheral blood of cancer patients, the amount of genomic DNA extracted from CTCs is extremely low, and using conventional amplification methods to detect single nucleotide mutations can easily lead to false negative results.

[0006] To solve the above problems, the present application adopts the following technical solutions: Basic scheme: a method for detecting lung cancer L858R mutation based on CRISPR / Cas9 and DNA double loop, comprising the following steps: Step one, enrich circulating tumor cells CTCs from blood samples; Step two, extract genomic DNA of the enriched CTCs; Step three, the DNA fragment containing L858R mutation is amplified by using recombinase polymerase amplification (RPA); Step four, the amplified DNA fragment is hybridized with the recognition loop in the DNA double loop structure, the DNA double loop is composed of the recognition loop and the reporter loop, and the partial region of the recognition loop is hybridized with the corresponding fragment of the reporter loop to form an overlapping double-stranded region; Step five, when the L858R mutation exists, the mutant DNA hybridizes with the recognition loop to activate the CRISPR / Cas9 system, under the guidance of sgRNA, Cas9 cuts the hybridized double-strand to release the reporter loop; wherein the sgRNA is designed for the L858R mutation site, the L858R mutation site is located at 12nt of the PAM sequence, a single nucleotide mismatch is introduced at the target sequence -1 to -3 relative to the L858R mutation target site at the distal end of the PAM site; and the 10-12nt near the PAM is the key seed region of Cas9 cutting activity, wherein the specificity is the strongest in the 8nt seed region; Step six, the released reporter loop is used as a template for rolling circle amplification (RCA) under the action of DNA polymerase; Step seven, the RCA amplification product is detected by using a fluorescent probe, and whether the L858R mutation exists is judged according to the fluorescence signal.

[0007] Beneficial effects: the present application covers the complete process from sample processing to final detection, solves the problem of extremely low content of CTC in blood by enriching CTC, improves the abundance of target DNA fragments by RPA amplification, realizes specific recognition and cutting of L858R mutation by combining DNA double loop with CRISPR / Cas9 system, releases the reporter loop to start RCA for signal amplification, finally detects by using a fluorescent probe, the whole process is coherent and efficient, and sensitive detection of lung cancer L858R mutation can be realized.

[0008] Preferably, the preparation method of the DNA double loop is: mixing the recognition loop single-stranded oligonucleotide and the circularization primer at a fixed molar ratio of 5:8, heating and slowly cooling, adding T4 DNA ligase for circularization reaction to form the recognition loop; then adding the reporter loop single-stranded oligonucleotide, repeating the heating, cooling and ligation reaction steps to form the DNA double loop with the interlocking double-stranded structure.

[0009] Beneficial effects: The preparation process of the DNA double ring is described in detail. By precisely controlling the conditions of each step, this method can stably prepare DNA double rings with specific structures, ensuring the structural stability and functional effectiveness of the DNA double ring in the subsequent detection process, laying the foundation for specific recognition and signal triggering of L858R mutations.

[0010] Preferably, the Cas9 enzyme and sgRNA are incubated at 37°C for 1 hour before the Cas9-sgRNA cleavage reaction to form a Cas9-sgRNA complex.

[0011] Beneficial effects: The formation conditions of the Cas9-sgRNA complex are determined. Incubation under these conditions can allow the Cas9 enzyme and sgRNA to fully bind and form a stable and active complex, thereby improving the efficiency of subsequent target DNA recognition and cleavage, ensuring the accuracy and reliability of the detection method.

[0012] Preferably, the Cas9 enzyme and sgRNA are incubated at 37°C for 1 hour before the Cas9-sgRNA cleavage reaction to form a Cas9-sgRNA complex.

[0013] Preferably, in the RPA amplification reaction, after the primers and DNA are added to the TwistAmp® Basic reaction mixture, MgOAc is added to initiate the reaction, and incubation is performed at 39°C for 20 minutes.

[0014] Beneficial effects: The specific reaction conditions for RPA amplification are determined. Under these conditions, the DNA fragment containing the L858R mutation can be efficiently amplified, providing sufficient target DNA for subsequent detection, improving the sensitivity of the detection, and avoiding complex temperature cycling process, simplifying the experimental operation.

[0015] Preferably, in the RCA reaction, the concentration of phi29 DNA polymerase is 30 U / μL, and the reaction time is 1 hour.

[0016] Beneficial effects: The optimized RCA reaction conditions can allow the reporter ring to efficiently undergo rolling circle amplification under the action of DNA polymerase, producing a large amount of amplification product, enhancing the fluorescence detection signal, and improving the detection ability of the detection method for low-abundance L858R mutations, ensuring accurate detection of mutations in very small amounts of samples.

[0017] Preferably, the fluorescent probe is composed of one DNA chain labeled with a fluorophore and another chain labeled with a quencher, and the concentration of the fluorescent probe is 300 nM.

[0018] Beneficial effects: The composition and concentration of the fluorescent probe are determined, at which the fluorescent probe can be fully hybridized with the RCA amplification product, and due to the design of the fluorophore and quencher, the fluorescent signal is quenched when not hybridized with the amplification product, and the fluorescent signal is restored after hybridization, which can produce obvious and accurate fluorescent signal changes, facilitating the detection and judgment of the presence of L858R mutation, and improving the accuracy and sensitivity of the detection.

[0019] Preferably, the ratio of Cas9 to sgRNA is 2:1.

[0020] Beneficial effects: At this ratio, Cas9 can fully bind to sgRNA, ensuring that almost all sgRNA molecules can form active complexes with Cas9, minimizing the degradation or non-specific interaction of sgRNA, thereby improving the cutting activity of Cas9 on target DNA, enhancing the recognition and cutting efficiency of the entire detection system for L858R mutation, and improving the accuracy of the detection.

[0021] Preferably, the cutting time of Cas9 is 15 minutes.

[0022] Beneficial effects: The optimized cutting time ensures that Cas9 can fully cut the target DNA, while avoiding the problems of increased non-specific cutting caused by prolonged cutting time, improving the specificity of the detection, accurately releasing the reporter loop, initiating the subsequent RCA reaction and fluorescence detection, and ensuring the reliability of the detection results.

[0023] Preferably, the method for enriching CTCs from blood samples is: using RosetteSep™ CTC enrichment cocktail containing anti-CD36 antibody, incubating the sample with the antibody mixture, and then centrifuging on a density gradient medium to collect the CTCs in the resulting cell layer.

[0024] Beneficial effects: This enrichment method uses specific antibodies to bind to CTC surface markers and combines with density gradient centrifugation technology, which can efficiently enrich CTCs from blood samples, improve the purity and quantity of CTCs, and provide a guarantee for subsequent extraction of high-quality genomic DNA from CTCs and accurate detection of L858R mutation, effectively solving the problem of low CTC content in blood and difficult detection.

[0025] Preferably, the Cas9-sgRNA system is resistant to single base mismatch, can maintain R-loop structure stability and effectively cut, and the sensitivity to two or three consecutive base mismatches introduced at the distal end of the PAM site is significantly improved. Continuous mismatches will reduce the thermodynamic stability of RNA-DNA hybridization, hinder the formation of R-loop, and significantly reduce or completely eliminate the cutting efficiency of Cas9. Single base mismatches at positions +1 and +2 distal to PAM are outside the critical discrimination range of Cas9, and do not significantly change the cutting efficiency of Cas9.

[0026] Beneficial effects: By precisely regulating the response of the Cas9-sgRNA system to different mismatches, specific differentiation of L858R single base mutation is achieved. The core advantage is that the resistance to single base mismatch ensures that the mutant sequence can form a stable R-loop and be efficiently cut, while the high sensitivity to two to three consecutive mismatches can significantly inhibit the cutting of wild type or other multi-base mutant sequences, forming a clear discrimination threshold. At the same time, the low sensitivity to single base mismatch in the non-key region distal to PAM improves the anti-interference ability to natural variation. Compared with traditional methods, the mutant and wild type can be distinguished by cutting efficiency difference without complex signal correction, simplifying the process while improving the detection specificity to single base resolution level, reducing external interference, and enhancing the detection stability and accuracy. It provides a reliable mechanism guarantee for efficient detection of lung cancer L858R mutation. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Schematic diagram for CRISPR / Cas9 and DNA double loop detection of EGFR L858R single nucleotide mutation.

[0028] Figure 2 Preparation and characterization of DNA double loop. A, schematic diagram of DNA double loop synthesis by T4 DNA ligase. B, PAGE analysis of DNA double loop synthesis process. Lane 1: 100 bp DNA ladder, lane 2: linear recognition loop, lane 3: recognition loop, lane 4: linear reporter loop, lane 5: linear reporter loop and recognition loop hybridization without T4 DNA ligase, lane 6: linear reporter loop and recognition loop hybridization with T4 DNA ligase, lane 7: DNA double loop with exonuclease I and III. C. AFM images of DNA double loop and recognition loop.

[0029] Figure 3Figure 1. DNA double circle map for Cas9 cleavage with L858R heteroduplex binding. A, Sequence alignment of MT and WT target, schematic of Cas9-mediated DNA cleavage. B, PAGE analysis of Cas9 cleavage of DNA double circle and RCA reaction in the presence of L858R. C, Fluorescence histogram of signal probe detection of RCA product. ****P<0.0001. D, Sequence alignment of mismatched DNA. E, PAGE analysis of single base, double base and triple base different mutations. F, Sequence alignment of different mutation positions. G, PAGE analysis of mutation sites including +1 / +2 positions. H, Quantitative analysis of Cas9 cleavage efficiency.

[0030] Figure 4 Figure 2. Optimization of experimental conditions. A. Concentration of phi29 DNA polymerase. B. RCA reaction time. C. Concentration of signal probe. D. Ratio of Cas9 to sgRNA. E. Cleavage time of Cas9. F. 15bp fluorescent probe with 4, 6, 8, 10, 12bp quencher probe. G. 18bp fluorescent probe with 4, 6, 8, 10, 12bp quencher probe. H. Fluorescence bar graph analysis of different mutation frequencies of L858R. I. Fluorescence bar graph analysis of different mutation types of EGFR.

[0031] Figure 5 Figure 3. Cell validation and spiked-in experiments. A. Sanger sequencing of RPA, amplification of genomic DNA of H3255 and A549 cell lines. B. Immunofluorescence images of 10 CTCs isolated from peripheral blood samples (scale bar: 50 μm). Green: anti-cytokeratin, red: anti-CD45, blue: DAPI. C. Fluorescence detection of H3255 cells spiked into whole blood samples (103, 102and 10 cells).

[0032] Figure 6 Figure 4. Detection of L858R in clinical serum samples. A. Fluorescence histogram of L858R and lung problem detection DNA of NSCLC patients. ***P<0.005. B. Fluorescence histogram of L858R patient and CTC detection of NSCLC patients. **P<0.05. DETAILED DESCRIPTION

[0033] The following will be further described in detail through specific embodiments: The method for detecting lung cancer L858R mutation based on CRISPR / Cas9 and DNA double circle of the present application comprises the following contents: 1. Sample preparation: 5 milliliters of peripheral blood sample is collected from a patient diagnosed with non-small cell lung adenocarcinoma. At the same time, peripheral blood of a healthy donor is collected as a control sample.

[0034] 2. CTC enrichment: The collected patient peripheral blood sample was mixed with RosetteSep™ CTC enrichment cocktail (containing anti-CD36 antibodies, etc.) and incubated.

[0035] The incubated sample was layered on lymphocyte separation medium (density: 1.077 g / mL) and centrifuged.

[0036] The cells in the cell layer obtained after centrifugation were collected, which were the enriched CTCs.

[0037] 3. DNA extraction: Genomic DNA was extracted from the enriched CTCs using a commercial kit (such as the kit from AG Biology). The specific steps are as follows: The enriched CTCs were lysed, and the lysate was transferred to a universal DNA mini column.

[0038] According to the DNA extraction kit protocol, the corresponding reagents were added in turn, and centrifugation was performed after each addition of reagent.

[0039] Finally, 50 μL of sterile water was added to elute the DNA, and the extracted DNA sample was stored at -20°C for standby.

[0040] 4. RPA amplification: According to the TwistAmp® DNA amplification kit. The extracted DNA and designed primers were added to the TwistAmp® Basic reaction mixture.

[0041] MgOAc was added to initiate the reaction, and the reaction was incubated at 39°C for 20 minutes to amplify the DNA fragments containing the L858R mutation. The amplification product was stored at -20°C.

[0042] 5. DNA double-loop preparation: The recognition loop single-stranded oligonucleotide (50 μM; 25 μL) and the circularization primer (50 μM; 40 μL) were mixed at a fixed molar ratio of 5:8.

[0043] The mixture was heated at 95°C for 5 minutes, and then slowly cooled to 25°C.

[0044] T4 DNA ligase (50U) was added, and the total activity of the added T4 DNA ligase was 50 units. The reaction was incubated at 37°C for 2 hours to perform the circularization reaction to form the recognition loop. After the reaction was completed, the enzyme was inactivated at 65°C for 10 minutes.

[0045] Add the reporter loop single-stranded oligonucleotide (50 μM), heat again at 95°C for 5 minutes, and slowly cool to 25°C.

[0046] Add T4 DNA ligase (50 U), incubate at 37°C for 2 hours to form a DNA double ring with an interlocked double-stranded structure.

[0047] 6. Preparation of Cas9-sgRNA complex: incubate Cas9 enzyme (1 μM) and sgRNA (1 μM) at 37°C for 1 hour to form a Cas9-sgRNA complex.

[0048] 7. Detection reaction: Hybridize the DNA double ring structure (40 μL) and the amplified DNA fragment containing the L858R mutation by heating at 95°C for 5 minutes and then gradually cooling to 25°C.

[0049] Add the Cas9-sgRNA complex to the hybridization mixture to initiate target recognition and cleavage reactions.

[0050] Perform RCA reaction with a phi29 DNA polymerase concentration of 30 U / μL and a reaction time of 1 hour.

[0051] Prepare signal probe: heat the Cy5 (10 μM) labeled fluorescent probe and the BHQ2 (10 μM) labeled quencher probe at 95°C for 5 minutes, and slowly cool to 25°C to form a signal probe.

[0052] Add the signal probe to the RCA product, incubate at 37°C for 20 minutes, and then perform fluorescence detection using a fluorescence spectrophotometer (Agilent Cary Eclipse).

[0053] 8. Result judgment: determine whether the L858R mutation exists based on the fluorescence intensity detected by the fluorescence spectrophotometer. If the fluorescence intensity is higher than the set threshold, it is determined that the L858R mutation exists in the sample; if it is lower than the threshold, it is determined that there is no mutation.

[0054] Comparison experiment and results: 1. Sensitivity comparison: Prepare a series of DNA samples containing different mutation frequencies (set multiple gradients from high to low), and use the method of the present application and the traditional qPCR method for detection.

[0055] The results show that the method of the present application can detect a mutation frequency as low as 1.0%, while the qPCR method is unstable when the mutation frequency is lower than 5.0%, and a large number of false negative results are obtained. This indicates that the method of the present application has higher sensitivity in detecting low-abundance L858R mutations.

[0056] 2. Specificity comparison: Using DNA samples containing common EGFR mutations (such as T790M and exon 19 deletion mutations) and L858R mutations, the present application method and the nucleic acid probe hybridization-based method were used for detection, respectively.

[0057] The method of the present application can accurately identify L858R mutations with high specificity, and almost no fluorescence signal is generated for other mutation type samples. The nucleic acid probe hybridization-based method has poor discrimination ability for different mutation types, and obvious cross-reaction occurs, with high misjudgment rate. This proves that the method of the present application has stronger specificity.

[0058] 3. Comparison of detection time: The same batch of samples containing L858R mutations were detected by the method of the present application and NGS technology, respectively.

[0059] The method of the present application can complete the whole process from sample collection to detection result within 6 hours, while NGS technology needs at least 11 hours, not including subsequent data processing and analysis time. This fully embodies the advantage of fast detection speed of the method of the present application.

[0060] From the above comparison experiment results, it can be seen that the method of the present application is obviously superior to the traditional detection method in sensitivity, specificity and detection time, and has creativity and non-obviousness.

[0061] The differences and advantages of the present application compared with the prior art such as the comparison file uploaded in the annex: Compared with the prior art, the present application has the following significant differences and advantages: 1、Technical combination innovation: The present application innovatively combines the CRISPR / Cas9 system with the DNA double-loop structure for lung cancer L858R mutation detection. In the prior art, although the CRISPR / Cas9 system and the DNA nanostructure have respective applications, there are few studies on the precise combination of the two and the use thereof for lung cancer L858R mutation detection. This unique technical combination enables the recognition loop in the DNA double-loop structure to specifically hybridize with the L858R mutation region during the detection process, forming a complementary double strand, which is then precisely recognized and cut by the Cas9-sgRNA complex, releasing the reporter loop and starting the subsequent signal amplification process, thereby greatly improving the specificity and sensitivity of the detection.

[0062] 2、Sample processing and signal amplification advantages: In terms of sample processing, the negative selection enrichment of circulating tumor cells (CTCs) effectively overcomes the problem of extremely low content of CTCs in blood, improves the purity and quantity of target cells, and provides a more reliable sample basis for subsequent detection. At the same time, the recombinase polymerase amplification (RPA) is used to amplify the DNA fragments containing the L858R mutation, obtaining abundant mutant DNA fragments, which solves the problem of extremely low amount of genomic DNA extracted from CTCs. In terms of signal amplification, the released reporter loop is used as a template for rolling circle amplification (RCA), achieving multi-stage amplification of the signal, which can detect a mutation frequency as low as 1.0%. The existing technologies such as qPCR have insufficient sensitivity in detecting low-abundance mutations, and the NGS technology, although theoretically sensitive, is complex to operate and costly.

[0063] 3、Detection performance improvement: The present application method is superior to traditional detection methods in terms of specificity, sensitivity and detection time. In terms of specificity, it can accurately distinguish L858R mutation from other common EGFR mutations, avoiding misjudgment caused by cross-reaction; in terms of sensitivity, it can detect low-abundance mutations, The specific implementation process is as follows: I、Experimental materials and reagents 1.1 Sample source Clinical samples: 5mL of peripheral blood from patients diagnosed with non-small cell lung cancer (NSCLC) in the University City Hospital of Chongqing Medical University from March to June 2025 (3 cases of L858R mutation positive, 10 cases of wild type), and 5mL of peripheral blood from healthy donors (negative control), all approved by the ethics committee (LL-202259).

[0064] Cell lines: H3255 cells carrying EGFR L858R mutation (positive control), A549 cells with EGFR wild type (negative control) were cultured in RPMI 1640 medium containing 10% FBS.

[0065] 1.2 Core reagents and instruments The sequences used in the present application are shown in Table 1: Table 1

[0066] DNA double loop preparation reagents: recognition loop single strand (50 μM; 25 μL), reporter loop single strand (50 μM), circularization primer (50 μM; 40 μL), T4 DNA ligase (50 U / μL, Sangon Biotech), 10% non-denaturing PAGE kit (Sangon Biotech).

[0067] CRISPR / Cas9 system: EnGen®Spy-Cas9 NLS (NEB), sgRNA (targeting L858R flanking PAM sequence, 1 μM, synthesized by Genscript).

[0068] Amplification and detection reagents: TwistAmp®RPA kit (TwistDx), phi29 DNA polymerase (0.5 U / μL, Sangon Biotech), fluorescent probe (Cy5 labeled, sequence: 5'-Cy5-TTTTTTT-BHQ2-3'), quenched probe (BHQ2 labeled, 8bp, Sangon Biotech).

[0069] CTC enrichment and identification reagents: RosetteSep™ CTC enrichment cocktail (containing anti-CD36 antibody, StemCell), lymphocyte separation medium (density 1.077 g / mL, Biosharp), anti-cytokeratin (CK)-FITC, anti-CD45-PE, DAPI (Abcam).

[0070] Instruments: Fluorescence spectrophotometer (Agilent Cary Eclipse), atomic force microscope (AFM, Bruker), fluorescence microscope (Olympus).

[0071] II. Experimental methods 2.1 Preparation and characterization of DNA double loop (corresponding to Figure 2) Cognate loop preparation: mix cognate loop single strand (50 mM; 25 pL) with circularized primer (50 mM; 40 pL) at 1 : 1.2 molar ratio, heat at 95 °C for 5 min, cool down slowly to 25 °C; add T4 DNA ligase (50 U), incubate at 37 °C for 2 h, deactivate at 65 °C for 10 min, form closed cognate loop.

[0072] Bi-cycle assembly: add reporter loop single strand (50 mM, complementary to cognate loop part), heat at 95 °C for 5 min, cool down slowly to 25 °C; add T4 DNA ligase (50 U) again, incubate at 37 °C for 2 h, deactivate at 65 °C for 10 min, form interlocked bi-cycle structure.

[0073] Verification: 10% non-denaturing PAGE analysis (Fig. 2B), where lane 6 shows bi-cycle specific band; AFM imaging (Fig. 2C) shows bi-cycle structure with diameter of about 200 nm, verifying successful assembly.

[0074] 2.2 Enrichment and identification of circulating tumor cells (CTCs) (corresponding to Fig. 5B) Enrichment: take 5 mL of patient peripheral blood, add RosetteSep™ antibody cocktail, incubate at room temperature for 20 min; layer on lymphocyte separation medium, centrifuge at 400 x g for 30 min, collect intermediate buffy coat layer (containing CTCs).

[0075] Identification: fix CTCs with 4% paraformaldehyde for 15 min, permeabilize with 0.2% Triton X-100 for 10 min, block with 5% BSA for 30 min; add anti-CK-FITC (green) and anti-CD45-PE (red), incubate at room temperature for 60 min, stain nuclei with DAPI (blue); screen CTCs (DAPI+ / CK+ / CD45-, Fig. 5B) under fluorescence microscope, count and collect.

[0076] 2.3 Genomic DNA extraction and RPA amplification DNA extraction: extract genomic DNA from enriched CTCs (AG Biology kit), quantify by Qubit (about 5-20 ng).

[0077] RPA amplification: use extracted DNA as template, use L858R-specific primers (forward: 5'-AGATCACAGATTTTGGGC-3', reverse: 5'-TTTGCCTCCTTCTGCATG-3'), follow TwistAmp® kit operation: incubate at 39 °C for 20 min, amplify 150 bp fragment containing L858R.

[0078] 2.4 CRISPR / Cas9 Cutting and RCA Signal Amplification (corresponding to Figures 1 and 3) Preparation of Cas9-sgRNA complex: Cas9 (final concentration 1 μM) and sgRNA (final concentration 2 μM) were incubated at 37°C for 1 h to form the optimal 2:1 complex (Figure 4D is a comparison of the optimization results).

[0079] Double-loop cleavage: The RPA product and DNA double loop (40 μL) were heated at 95 °C for 5 min and cooled to 25 °C; the Cas9-sgRNA complex was added and cleaved at 37 °C for 15 min (Figure 4E optimization results), releasing the reporter loop.

[0080] RCA amplification: Add phi29 DNA polymerase (0.5 U / μL, Figure 4A Optimization result) and dNTP (0.6 μM), incubate at 30℃ for 1 h (Figure 4B Optimization result), and the reporter loop is amplified into a long repeat sequence.

[0081] The principle of CRISPR / Cas9 DNA double-loop detection of L858R single-base mutations is as follows: Figure 1 As shown. When cDNA containing the EGFR L858R mutation is present in solution, the recognition sequence within the DNA double loop hybridizes with the L858R sequence to form a complementary double strand. The sgRNA in the CRISPR / Cas9-sgRNA complex then recognizes the PAM site within the double strand and activates Cas9 to cleave the DNA three nucleotides upstream of the PAM site. After cleavage, the reporter loop is released from the DNA double loop structure and serves as a template for the subsequent RCA reaction. A signal probe consisting of one DNA strand labeled with a quencher and another strand labeled with a fluorophore is added to the RCA reaction mixture. The RCA product competitively binds to the fluorescent probe, displacing it from the quencher strand. Upon hybridization with the RCA product, the fluorescent probe dissociates from the quencher, resulting in the recovery of the fluorescence signal. The fluorescence signal is detected using a fluorescence spectrophotometer at a specific excitation wavelength. This mutation-specific release of the reporter loop for amplification occurs only in the presence of the mutated ctDNA. Other drug-resistant mutations in patients with non-small cell lung adenocarcinoma do not trigger loop release, therefore no amplification occurs and no fluorescence signal is detected.

[0082] like Figure 3 As shown, the DNA double loop recognizes L858R and triggers Cas9-mediated cleavage. First, the ability of the DNA double loop to recognize L858R and the cleavage activity of Cas9 were evaluated. Intermediate and final products were analyzed by 8% PAGE. The DNA double loop was incubated with L858R and WT sequences, respectively. Figure 3A). It was observed that both DNA double loops and Cas9 can hybridize with recognition loops ( Figure 3 Lanes B, 3, and 5 indicate that single-base mutations do not significantly affect double-strand formation. After adding the Cas9-sgRNA complex and incubating at 37°C, both WT and L858R sequences were recognized and cleaved by Cas9 at 20°C (lanes 4 and 6), ultimately leading to the formation of RCA products (lanes 7 and 8). Signal probes were added to both RCA products, and fluorescence signals were measured using a fluorescence spectrophotometer. The results show that although both WT and L858R trigger Cas9-mediated cleavage followed by rolling circle amplification, the fluorescence intensity of the WT group was significantly lower than that of the L858R group (lanes 3 and 5). Figure 3 C). Although CRISPR-Cas9 is known for its high target specificity, its recognition of DNA-sgRNA double strands is not absolutely stringent. In other words, it exhibits some tolerance for single-base mismatches. Notably, the L858R mutation site is 12 nt away from the PAM sequence, and Cas9 is generally more tolerant of mismatches there. While a single base mismatch at this location does not completely eliminate Cas9's cleavage activity, it does lead to a decrease in the efficiency of target fragment cleavage.

[0083] These results led us to question the specificity of Cas9 for single-base mutations and prompted us to conduct further research and validation. We introduced two-base and three-base mismatches at the original mutation site located distal to the PAM site. Figure 3 D). PAGE analysis ( Figure 3 The results (EF) indicate that the ability of Cas9 to recognize and cleave DNA double loops gradually decreases with increasing number of mutated bases. Notably, this change is significantly amplified in the presence of three nucleotide mismatches. The Cas9-sgRNA system exhibits a degree of mismatch tolerance during target DNA recognition. The R-loop structure remains stable in the presence of a single base mismatch, allowing Cas9 to efficiently cleave the target. However, the thermodynamic stability of RNA-DNA hybridization is significantly reduced in the presence of two to three consecutive mismatches, hindering the formation of the R-loop structure. As a result, Cas9 cleavage efficiency is significantly reduced or completely eliminated. Previous studies have shown that two base mismatches near the proximal end of the PAM sequence significantly reduce Cas9 activity, and three consecutive mismatches further amplify this drawback, consistent with our results. Although the two base pair mismatches introduced in our study are located at positions 12–13 nt in PAM (a region relatively less sensitive to mismatches), they still produced a measurable (albeit modest) reduction in Cas9 cleavage efficiency. This further supports Cas9's good tolerance to mismatches in the distal region of PAM.

[0084] To further evaluate the recognition and cleavage efficiency of Cas9, we designed single base mismatches at positions +1 and +2 distal to the PAM site Figure 3 G). As shown in Figs. Figure 3 H-I, the position of the mismatch did not significantly change the cleavage efficiency of Cas9. This indicates that the introduced mismatch is outside the critical discrimination range of Cas9, which is generally across the PAM 12 nt with the strongest specificity within the 8 nt seed region. According to previous studies, the 10-12 nt proximal to the PAM has been identified as the critical seed region for Cas9 cleavage activity, and mismatches within the 10-12 nt range significantly reduced the editing efficiency of Cas9. The L858R mutation site is located at 12 nt from the PAM sequence. Therefore, single nucleotide mismatches at positions -1 to -3 (relative to the target site) enable precise single base discrimination.

[0085] To improve the performance of the method, the key experimental parameters were systematically optimized as shown in Figs. Figure 4 including the Cas9 cleavage time, Cas9 to sgRNA reaction ratio, signal probe concentration, and RCA reaction time.

[0086] First, the concentration of phi 29 DNA polymerase was optimized (Fig. Figure 4 A). The reaction reached saturation at 30 °C with the addition of U / μL of DNA polymerase. Next, the RCA reaction time was optimized (Fig. Figure 4 B). The maximum fluorescence intensity was observed when the RCA reaction time was set to 1 hour. This can be due to the increasing complexity of the RCA product structure at later stages, which can hinder probe binding due to steric effects. The concentration of the signal probe was also optimized. The results showed that the final probe concentration of 300 nM had the highest detection efficiency in a 200 μL detection system (Fig. Figure 4 C). Subsequently, the Cas9 to sgRNA ratio was optimized. The highest cleavage efficiency was achieved when the Cas9:sgRNA ratio was 2:1 (Fig. Figure 4 D). Excess Cas9 can ensure that almost all sgRNA molecules are fully bound and minimize degradation or non-specific interactions, thereby maximizing cleavage activity and fluorescence output. In contrast, when the amount of Cas9 is excessive but sgRNA is insufficient, a large amount of unloaded Cas9 protein can occupy the target site without cleavage, leading to competitive inhibition, which impairs the formation of functional complexes. Finally, the cleavage time of Cas9 was optimized, and the results showed that the highest cleavage efficiency and maximum fluorescence intensity were achieved at 15 minutes (Fig. Figure 4 E).

[0087] We optimized the length and ratio of the signal probe. Initially, two 15 bp and 18 bp fluorescent probes were designed and paired with different lengths (4 bp, 6 bp, 8 bp, 10 bp, and 12 bp) of quencher probes. The experimental results showed that the fluorescence intensity gradually decreased with the increase of the length of the quencher probe. Notably, the 15 bp fluorescent probe showed the smallest background signal (F) when paired with the 8 bp quencher probe, while the 18 bp fluorescent probe achieved the best background suppression (G) when combined with the 10 bp quencher probe. Figure 4 Figure 4

[0088] Therefore, in subsequent experiments, the following optimized conditions were adopted: 30 U / μL phi29 DNA polymerase, 300 nM signal probe, Cas9:sgRNA ratio of 2:1, and cutting time of 15 minutes.

[0089] 2.5 Fluorescence detection and result analysis Signal detection: Add fluorescent probe (300 nM, optimized results in Figure 4C) and quencher probe (8 bp), incubate at 37°C for 20 min; detect Cy5 signal (excitation 649 nm, emission 670 nm) by fluorescence spectrophotometer.

[0090] Criteria: Mutant group fluorescence intensity > wild type mean + 3 times standard deviation is positive.

[0091] Three, experimental results 3.1 Specific cutting of DNA double circle (corresponding to Figure 3) PAGE analysis shows (Figure 3B): In the L858R mutant sample, Cas9-sgRNA cuts the double circle to release the reporter circle (lane 6), and the wild type sample has significantly reduced cutting efficiency (lane 4); in the RCA product (lanes 7-8), the mutant group yield is 3.2 times that of the wild type.

[0092] Fluorescence results (Figure 3C): The fluorescence intensity of the mutant group is significantly higher than that of the wild type.

[0093] 3.2 Detection condition optimization (corresponding to Figure 4) Key parameters: phi29 concentration 0.5 U / μL, RCA time 1 h, fluorescent probe 300 nM, Cas9:sgRNA = 2:1, cutting time 15 min, mutant / wild type fluorescence ratio (SNR) is the highest (Figure 4A-E).

[0094] Probe optimization: 15 bp fluorescent probe + 8 bp quencher probe combination has the lowest background (Figure 4F), with a signal-to-noise ratio of 4.8. ​​

[0095] 3.3 Sensitivity and specificity (corresponding to FIG. 4H, 4I) Sensitivity: Detection of serially diluted H3255 / A549 mixed DNA (mutation frequency 0.1-10%) showed that the method could stably detect as low as 1.0% mutation frequency (FIG. 4H), which was superior to traditional qPCR (detection limit 5.0%).

[0096] Specificity: No cross-reactivity to other EGFR mutations (T790M, exon 19 deletion) (FIG. 4I), 100% specificity.

[0097] Under the optimized experimental conditions, we evaluated the sensitivity and specificity of the CRISPR / Cas9-based DNA duplex detection system. First, to evaluate the sensitivity, we prepared DNA samples with different mutation frequencies. As shown in FIG. 4H, the system could detect the L858R mutation at a frequency as low as 1.0%. Figure 4 H.

[0098] Although the sensitivity of our method was slightly lower than previously reported methods, it is worth noting that these studies only used tissue samples and ctDNA for detection. Certain advanced techniques, such as digital PCR (dPCR) and NGS, can theoretically detect mutation frequencies below 0.1%, but these methods usually require complex instruments, high sequencing depth, or expensive reagents. In contrast, our method achieved comparable sensitivity through a simplified workflow and minimal equipment requirements. It is worth noting that in CTC-based detection, the amount of genomic DNA is extremely limited, and a mutation frequency threshold of 1.0% is clinically acceptable. Therefore, our system can meet the needs of early diagnosis and treatment monitoring of non-small cell lung cancer in real clinical applications. Next, we tested the specificity of the system using common EGFR mutations, including T790M and exon 19 deletion mutations. The results showed that the CRISPR / Cas9-mediated DNA duplex detection system could accurately identify the L858R mutation with high specificity (FIG. 4I). Figure 4 I.

[0099] 3.4 CTC Spiking experiment (corresponding to FIG. 5C) H3255 cells (0, 1, 2, 5, 10) were added to the peripheral blood of healthy people , , and after enrichment, the fluorescence intensity decreased with the decrease of cell number, but the 10 cell group was still significantly higher than the wild type (P<0.01, FIG. 5C), indicating that mutations as low as 2 CTC / mL blood could be detected.

[0100] 3.5 Clinical sample verification (corresponding to FIG. 6) 3 L858R positive patients CTC samples were all positive (fluorescence intensity 3890±412 AU), 10 wild type patients were all negative (1120±185 AU, P<0.05, Fig. 6B), consistent with NGS results 100%.

[0101] The embodiment realizes the ultra-sensitive detection of EGFR L858R mutation in CTC (1.0% mutation frequency) through the process of "CTC enrichment→DNA double loop recognition→Cas9 cutting release report loop→RCA amplification→fluorescence detection", which is significantly superior to the prior art in specificity and clinical applicability, and fully embodies the breakthrough of CRISPR / Cas9 combined with DNA double loop.

[0102] The above is only an embodiment of the present application, and the specific technical solutions and / or common knowledge of the scheme are not described in detail. It should be pointed out that for those skilled in the art, without departing from the technical scheme of the present application, a number of modifications and improvements can be made, which should also be considered as the protection scope of the present application, which will not affect the effect and practicality of the present application. The protection scope of the present application should be subject to the content of its claims, and the specific implementation mode and the like recorded in the specification can be used to explain the content of the claims.

Claims

1. A method for detecting lung cancer L858R mutation based on CRISPR / Cas9 and DNA double circle, characterized in that, The method comprises the following steps: Step 1: enriching circulating tumor cells (CTCs) from a blood sample; Step 2: extracting genomic DNA of the enriched CTCs; Step 3: amplifying the DNA fragment containing the L858R mutation by recombinase polymerase amplification (RPA); Step 4: hybridizing the amplified DNA fragment with a recognition loop in a DNA double loop structure, the DNA double loop being composed of a recognition loop and a reporter loop, and a partial region of the recognition loop being hybridized with a corresponding fragment of the reporter loop to form an overlapping double-stranded region; Step 5: when the L858R mutation exists, the mutated DNA hybridizes with the recognition loop to activate the CRISPR / Cas9 system, and under the guidance of sgRNA, Cas9 cuts the hybridized double strand to release the reporter loop; wherein the sgRNA is designed for the L858R mutation site, the L858R mutation site is located at 12nt of the PAM sequence, a single nucleotide mismatch is introduced at positions 1 to 3 of the target sequence relative to the L858R mutation target site at the distal end of the PAM site, and the 10-12nt at the proximal end of the PAM is a key seed region for Cas9 cutting activity, wherein the specificity is the strongest in the 8nt seed region; Step 6: performing rolling circle amplification (RCA) on the released reporter loop as a template under the action of DNA polymerase; Step 7: detecting the RCA amplification product using a fluorescent probe, and determining whether the L858R mutation exists according to the fluorescence signal.

2. The method for detecting lung cancer L858R mutation based on CRISPR / Cas9 and DNA double loop according to claim 1, characterized in that, The preparation method of the DNA double loop is as follows: mixing the recognition loop single-stranded oligonucleotide and the circularization primer at a fixed molar ratio of 5:8, heating, slowly cooling, adding T4 DNA ligase for circularization reaction to form the recognition loop, then adding the reporter loop single-stranded oligonucleotide, repeating the heating, cooling and ligation reaction steps to form the DNA double loop with an interlocking double-stranded structure.

3. The method of claim 1, wherein, Before performing the Cas9-sgRNA cutting reaction, the Cas9 enzyme and the sgRNA are incubated at 37°C for 1 hour to form a Cas9-sgRNA complex.

4. The method of claim 1, wherein, In the RPA amplification reaction, the primers and DNA are added to the TwistAmp®Basic reaction mixture, the reaction is initiated by adding MgOAc, and incubation is performed at 39°C for 20 minutes.

5. The method of claim 1, wherein, In the RCA reaction, the concentration of phi29 DNA polymerase is 30 U / μL, and the reaction time is 1 hour.

6. The method of claim 1, wherein, The fluorescent probe is composed of one DNA strand labeled with a fluorophore and the other strand labeled with a quencher, and the concentration of the fluorescent probe is 300 nM.

7. The method of claim 1, wherein, The ratio of Cas9 to sgRNA is 2:

1.

8. The method of claim 1, wherein, The cutting time of Cas9 is 15 minutes.

9. The method of claim 1, wherein, The method for enriching CTCs from a blood sample is: using RosetteSepTM CTC enrichment cocktail containing anti-CD36 antibody, after the sample is incubated with the antibody mixture, centrifugation is carried out on a density gradient medium, and CTCs in the obtained cell layer are collected.

10. The method of claim 1, wherein, The Cas9-sgRNA system is resistant to single base mismatch, can maintain R-loop structure stability and effectively cut, and the sensitivity to two or three consecutive base mismatches introduced far from the PAM site is significantly improved. Continuous mismatch will reduce the thermodynamic stability of RNA-DNA hybridization, hinder the formation of R-loop, and significantly reduce or completely eliminate the cutting efficiency of Cas9; the single base mismatch at the +1 and +2 positions far from the PAM has no significant effect on the cutting efficiency of Cas9.

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