DNA flap controlled FCT-CRISPR detection system and application

Abstract

The invention relates to the technical field of biological detection, in particular to a DNA skin flap controlled FCT-CRISPR detection system and application. The FCT-CRISPR detection system comprises a dumbbell probe containing a DNA skin flap structure, a CRISPR / Cas12a protein, crRNA, a reporter probe and a Cas12a splitting activator SA1, and the dumbbell probe is a dumbbell probe containing a DNA skin flap structure. The DNA skin flap structure comprises a 5 '-end skin flap and a 3'-end skin flap, the 5 '-end skin flap is a Cas12a split activator SA2, and the 5'-end skin flap can be recognized and cut by skin flap endonuclease 1. The detection reaction process adopting the FCT-CRISPR detection system does not need to depend on an additional DNA connection or replication process, and sensitive, accurate and mixed read-and-read detection of the FEN1 activity can be realized.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and in particular to a DNA flap-controlled FCT-CRISPR detection system and its application. Background Technology

[0002] Flap endonuclease 1 (FEN1) is a highly conserved structure-specific nuclease that plays a crucial role in DNA replication and repair pathways, essential for maintaining genome integrity. In addition to its typical flap cleavage activity, FEN1 is involved in telomere maintenance, resolution of stalled replication forks, and apoptosis. Dysregulation of FEN1 activity is closely associated with genomic instability and is linked to various cancers, such as breast, gastric, and ovarian cancer, often with poor prognosis and aggressive tumor phenotypes. Therefore, FEN1 has become a promising biomarker for cancer diagnosis and prognosis, as well as a potential therapeutic target, driving the development of inhibitors such as myricetin and SC13. These characteristics highlight the urgent need for accurate, sensitive, and easily operable methods to quantify FEN1 activity in biomedical research and clinical settings.

[0003] Traditional methods for detecting FEN1 activity, including gel electrophoresis, enzyme-linked immunosorbent assay (ELISA), and Western blotting, primarily rely on indirect measurements of cleavage products or protein abundance. These methods typically have several limitations: gel-based methods are semi-quantitative, time-consuming, and lack sensitivity for detecting low-abundance samples; immunoassays require high-quality antibodies and cannot distinguish between active and inactive isoforms; and Western blotting only provides semi-quantitative data on FEN1 levels, not direct enzyme activity. Emerging fluorescence-based assays, employing molecular beacons or fluorophore-quencher-labeled oligonucleotide probes, can directly detect FEN1 activity and improve sensitivity, and are considered promising methods for FEN1 activity detection. In particular, various nucleic acid amplification methods, such as exponential amplification, rolling circle amplification, and hybridization chain reaction (LCR), have been integrated into fluorescence-based assays to enhance detection sensitivity. However, even these fluorescence-based assays still face significant challenges: they typically involve complex probe design, require multiple enzymatic steps, and are prone to nonspecific amplification and high background signals due to primer dimer-induced initiation, ultimately limiting their practical applicability in complex biological matrices.

[0004] In recent years, the integration of CRISPR / Cas systems into biosensor design has revolutionized the field of molecular detection, thanks to their programmability, extremely high specificity, and powerful trans-cleavage activity, enabling significant signal amplification. Cas12a, upon recognizing a specific activator DNA sequence, releases non-specific single-stranded DNA cleavage activity—a characteristic that makes it ideal for signal amplification in FEN1 activity detection. Several research groups have attempted to use CRISPR / Cas12a to detect FEN1 activity. However, these CRISPR-based strategies require additional enzymatic steps, such as DNA ligation (linking fragmented activators) or DNA polymerization (extending primers to complete activators), to generate functional CRISPR / Cas12a activator sequences after FEN1 action. The introduction of these auxiliary reactions not only increases operational complexity, cost, and assay time but also introduces the risk of false positive signals due to non-specific ligation, incorrect primer binding, or polymerase errors. These limitations hinder the reliability, simplicity, and widespread applicability of existing CRISPR-based strategies in direct enzyme activity monitoring. Therefore, a novel strategy is needed to address these limitations of existing CRISPR-based strategies. Summary of the Invention

[0005] To address the problems existing in the prior art, the purpose of this invention is to provide a DNA flap-controlled CRISPR / Cas12a detection system and its application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a DNA flap-controlled FCT-CRISPR detection system, comprising a dumbbell probe containing a DNA flap structure, a CRISPR / Cas12a protein, crRNA, a reporter probe, and a Cas12a cleavage activator SA1; The DNA flap structure includes a 5'-end flap and a 3'-end flap. The 5'-end flap is a Cas12a cleavage activator SA2, and the 5'-end flap can be recognized and cleaved by flap endonuclease 1.

[0007] A second aspect of the present invention provides a detection kit comprising the FCT-CRISPR detection system described herein.

[0008] A third aspect of the present invention provides the application of the FCT-CRISPR detection system or detection kit described herein in the detection of endonuclease 1 activity in skin flaps, wherein the application is not intended for the diagnosis or treatment of disease.

[0009] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The FCT-CRISPR detection system designed in this invention is a division activator system composed of Cas12a division activator 1 (SA1) and Cas12a division activator 2 (SA2), wherein SA2 is embedded in a dumbbell probe (a specific substrate of FEN1) as a 5'-end flap. When FEN1 recognizes and cleaves, SA2 is released from the dumbbell probe and synergistically activates the trans-cleavage activity of CRISPR / Cas12a with SA1, generating an amplified fluorescence signal. In the absence of FEN1, the steric hindrance of the dumbbell probe prevents SA2 from binding to CRISPR / Cas12a, effectively suppressing the background signal. This reaction process does not rely on additional DNA ligation or replication processes, thus achieving sensitive, accurate, and ready-to-read detection of FEN1 activity. Not only does it minimize the background signal through structural inhibition, but it also simplifies the assay to a single enzymatic step.

[0010] (2) The FCT-CRISPR detection system of this invention has high sensitivity and specificity, with a detection limit as low as 0.2 mU, and excellent specificity for non-target enzymes. In addition, the successful detection application in cancer cell lysates demonstrates the clinical application potential of FCT-CRISPR, which is easy to apply to inhibitor screening and the analysis of complex biological samples, and has strong translational application potential. Attached Figure Description

[0011] Figure 1 This is a schematic diagram of the FEN1 activity detection system controlled by the DNA flap in an embodiment of the present invention. Figure 2 This invention provides a feasibility verification of the FCT-CRISPR detection system in this embodiment. A shows a schematic diagram of CRISPR / Cas12a activation by different activators; B shows the real-time fluorescence spectrum of the FCT-CRISPR detection system under different activator conditions; C shows the gel electrophoresis diagram of the FCT-CRISPR detection system under different conditions; D shows the real-time fluorescence spectrum of the FCT-CRISPR detection system with and without FEN1; E shows a bar chart comparing the fluorescence intensity of the FCT-CRISPR detection system with and without FEN1; the Cas12a concentration is 25 nM, SA1 concentration is 200 nM, SA2 concentration is 200 nM, dumbbell probe concentration is 200 nM, reporter probe concentration is 1600 nM, the reaction temperature is 37℃, and the error bar represents the standard deviation of three experiments. Figure 3This study investigates the influence of various factors on the detection strategy. A shows a schematic diagram of different activator lengths; B shows a real-time fluorescence spectrum; C shows a comparison of fluorescence intensity for different activator lengths; D shows a schematic diagram of different activator intervals; E shows a real-time fluorescence spectrum; F shows a comparison of fluorescence intensity for different activator intervals; G shows a schematic diagram of different 3' end lengths of the dumbbell probe; H shows a real-time fluorescence spectrum; I shows a comparison of fluorescence intensity for different 3' end lengths of the dumbbell probe; J shows a schematic diagram of different dumbbell probe sizes; K shows a real-time fluorescence spectrum; L shows a comparison of fluorescence intensity for different dumbbell probe sizes; M shows a schematic diagram of different protrusion positions; N shows a real-time fluorescence spectrum; O shows a comparison of fluorescence intensity for different protrusion positions. The error bars in the figures represent the standard deviation of three experiments. Figure 4 These are real-time fluorescence spectra under different reporter probe types in embodiments of the present invention; Figure 5 This is a schematic diagram of Cas12a concentration optimization in an embodiment of the present invention; in the figure, A is a bar chart comparing the fluorescence intensity of the negative and positive systems under different Cas12a concentrations; B is a trend chart showing the change of the ratio of fluorescence intensity of the negative and positive systems under different Cas12a concentrations. Figure 6 This invention provides an example of dumbbell probe concentration optimization; wherein, A is a bar chart comparing the fluorescence intensity of the negative and positive systems under different dumbbell probe concentrations; B is a graph showing the trend of the ratio of fluorescence intensity of the negative and positive systems under different dumbbell probe concentrations. Figure 7 This is a schematic diagram illustrating the optimization of report probe concentration in an embodiment of the present invention; wherein, A is a bar chart comparing the fluorescence intensity of the negative and positive systems under different report probe concentrations; B is a graph showing the trend of the ratio of fluorescence intensity of the negative and positive systems under different report probe concentrations; Figure 8 This is an evaluation of the detection performance of the FCT-CRISPR detection system in this embodiment of the invention; wherein, A is the real-time fluorescence spectrum at different FEN1 enzyme concentrations; B is the relationship between FEN1 concentration and fluorescence intensity; C is the linear equation graph, the linear equation is F=14417C_FEN1+180.11, and the correlation coefficient (R) is... 2 The value is 0.993; D is the real-time fluorescence response spectrum for different samples, and E is the bar chart of fluorescence signal intensity; the error bars in the figure represent the standard deviation of the three experiments; Figure 9 This is an evaluation of the detection performance of the FCT-CRISPR detection system in actual samples in this embodiment of the invention; wherein, A is a schematic diagram of the detection principle; B is a real-time fluorescence response spectrum of different actual samples; C is a fluorescence intensity bar chart of different actual samples; the error bars in the figure represent the standard deviation of three experiments; Figure 10 This is a schematic diagram illustrating the linear relationship between cell count and fluorescence intensity detected by the FCT-CRISPR detection system in an embodiment of the present invention; the error bars in the figure represent the standard deviation of three experiments. Detailed Implementation

[0012] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0013] As mentioned earlier, existing CRISPR / Cas12a-based strategies for detecting FEN1 activity require additional enzymatic steps, such as DNA ligation (ligating fragmented activators) or DNA polymerization (extending primers into complete activators). The introduction of these auxiliary reactions not only increases operational complexity, cost, and assay time, but also introduces the risk of false positive signals due to non-specific ligation, incorrect primer binding, or polymerase errors. In view of this, the present invention proposes a direct, single-tube, and ready-to-read FEN1 activity detection strategy—the DNA flap-controlled CRISPR / Cas12a trans-cutting detection system (FCT-CRISPR).

[0014] In a typical embodiment of the present invention, a DNA flap-controlled FCT-CRISPR detection system is provided, comprising a dumbbell probe containing a DNA flap structure, a CRISPR / Cas12a protein, crRNA, a reporter probe, and a Cas12a cleavage activator SA1; the DNA flap structure includes a 5'-end flap and a 3'-end flap, the 5'-end flap being the Cas12a cleavage activator SA2, and the 5'-end flap being cleaved by flap endonuclease 1.

[0015] This invention designs a dumbbell probe with a DNA flap structure in the FCT-CRISPR detection system, wherein the 5'-end flap domain serves as the cleavage activator SA2 (a specific substrate for FEN1), and the dumbbell-shaped scaffold serves as a steric hindrance unit. The process of detecting FEN1 activity using the DNA flap-controlled FCT-CRISPR detection system is as follows: Figure 1 As shown, when FEN1 recognizes and cleaves the target cell, SA2 is released from the dumbbell probe and synergistically activates the trans-cleavage activity of CRISPR / Cas12a with SA1, generating an amplified fluorescence signal. In the absence of FEN1, the steric hindrance of the dumbbell probe prevents SA2 from binding to CRISPR / Cas12a, effectively suppressing the background signal.

[0016] In some embodiments, the stem length of the dumbbell probe is 6 to 18 nt.

[0017] In some embodiments, the dumbbell probe is positioned relative to the interior of the Cas12a protein.

[0018] In some embodiments, the length of the 3'-end flap is -1 to 5 nt, preferably 0 to 5 nt.

[0019] In this context, a 3'-end flap length of 0 means the 3'-end flap has no protruding bases, a 3'-end flap length of 1 means the 3'-end flap has one protruding base, and a 3'-end flap length of 5 means the 3'-end flap has five protruding bases. In some embodiments, the number of bases complementary to crRNA for SA1 and SA2 is the same, which is 8-9 nt.

[0020] In some embodiments, the FCT-CRISPR detection system also includes a buffer solution.

[0021] In some embodiments, after SA1 and SA2 bind to crRNA, the interval between SA1 and SA2 is 0~2nt, preferably 0nt.

[0022] The interval of 0 between SA1 and SA2 means that there is no spacer base between SA1 and SA2.

[0023] In some embodiments, the sequence of the dumbbell probe is as shown in SEQ ID NO: 1.

[0024] In some implementations, the sequence of SA1 is as shown in SEQ ID NO: 2.

[0025] In some implementations, the sequence of SA2 is as shown in SEQ ID NO: 3.

[0026] In some implementations, the crRNA sequence is shown in SEQ ID NO: 4.

[0027] In some implementations, the reporting probe is a linear or hairpin structure, preferably a linear structure.

[0028] In some implementations, when the reporting probe is a linear structure, its sequence is as shown in SEQ ID NO: 5; when the reporting probe is a hairpin structure, its sequence is as shown in SEQ ID NO: 6.

[0029] In another specific embodiment of the present invention, a detection kit is provided, which includes the FCT-CRISPR detection system described in the present invention.

[0030] In another specific embodiment of the present invention, the FCT-CRISPR detection system or detection kit of the present invention is used in the detection of FEN1 activity, and the application is not for the purpose of disease diagnosis and treatment.

[0031] In some embodiments of the present invention, detecting FEN1 activity includes the following steps: S1. Mix CRISPR / Cas12a protein and crRNA in buffer and incubate to obtain Cas12a / crRNA complex; S2. Mix the dumbbell probe, Cas12a cleavage activator SA1, Cas12a / crRNA complex, reporter probe and buffer to obtain the reaction system; S3. Add the skin flap endonuclease 1 sample to be tested into the reaction system, carry out the isothermal reaction, detect the generated fluorescence signal and analyze the results.

[0032] In some embodiments, in step S1, the CRISPR / Cas12a protein and crRNA are mixed in a buffer and incubated at 37°C for 30 min.

[0033] In some implementations, in step S1, the molar ratio of CRISPR / Cas12a protein to crRNA is 1:2.

[0034] In some embodiments, in step S1, the buffer solution contains DEPC water and RNase inhibitors.

[0035] In some embodiments, in step S2, the working concentration range of the dumbbell probe is 0.1~0.3 μM, the working concentration range of the Cas12a cleavage activator SA1 is 0.1~0.3 μM, the working concentration range of the Cas12a / crRNA complex is 20~30 nM, and the working concentration range of the reporter probe is 0.8~1.2 μM.

[0036] In some implementations, the isothermal reaction conditions are 25-37°C for 20-70 min.

[0037] In some implementations, the buffer solution is a CutSmart buffer solution.

[0038] The detailed information and sources of the chemical reagents and instruments involved in the following examples are as follows: 1×CutSmart buffer (50 mM potassium acetate, 20 mM Tris-acetic acid, 10 mM magnesium acetate, 100 µg / mL recombinant albumin, pH 7.9) and DL-dithiothreitol (DTT) were purchased from New England Biolabs (Beijing, China). Recombinant LbCas12a (CRISPR / Cas12a protein), 5×TBE buffer (2.0 mM EDTA, 89 mM borate, 89 mM Tris, pH 8.3), and RNase inhibitors were purchased from Sangon Biotech. Co., Ltd. (Shanghai, China). Ammonium persulfate (APS) and tetramethylethylenediamine (TEMED) were purchased from Beyotime Biotechnology (Shanghai, China). DNA markers and 6× loading buffer were purchased from Takara Bio Co., Ltd. (Beijing, China). The HPLC-purified oligonucleotides listed in Table 1 were synthesized by Sangon Biotech. Co., Ltd. (Shanghai, China). Other reagents were used directly without further purification. The reaction solution was prepared using water treated with diethyl pyrocarbonate (DEPC), purchased from Sangon Biotech.

[0039] Real-time fluorescence measurements were performed using an Infinite® M200 PRO microplate reader (TECAN, Switzerland). Gel electrophoresis experiments were conducted using a JY-SCZ9 electrophoresis tank, powered by an HX-105 constant temperature water circulation system and a DYY-6C electrophoresis power supply (both from Beijing Changliu Scientific Instruments Co., Ltd.). Gel imaging was performed using a Tanon 3500-BR UV imaging system (Shanghai Tianneng Life Science Co., Ltd.).

[0040] The actual sample preparation methods in the following examples are as follows: Cancer cells MCF-7 and HeLa were cultured in Dulbecco modified Eagle medium (DMEM). This medium was supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 mg / mL streptomycin. Cells were incubated at 37°C in a humidified atmosphere containing 5% carbon dioxide (CO2).

[0041] To prepare the cell extract, approximately 500,000 cultured cells were first collected and then lysed on ice for 30 minutes using RIPA lysis buffer (Sangon, China). After lysis, the sample was centrifuged at 12,000 rpm for 10 minutes to separate the supernatant, which was then stored at -20°C for later use.

[0042] Table 1. Nucleic acid sequences involved in the examples

[0043] The experimental methods involved in the following embodiments are as follows: The dumbbell probe and the splitting activator were annealed according to their respective protocols as follows. The dumbbell probe was incubated in 1×CutSmart buffer at 95°C for 5 minutes, and then slowly cooled to room temperature for later use. Splitting activator 1 (SA1) and splitting activator 2 (SA2) were annealed according to the same protocol as described above.

[0044] The Cas12a / crRNA complex was prepared prior to subsequent experiments as follows: 14 μL of diethyl pyrocarbonate (DEPC)-treated water, 1.0 μL of LRNase inhibitor (40 U / μL), 2.0 μL of crRNA (2.0 μM), and 2.0 μL of Cas12a (1.0 μM) were mixed in 1×CutSmart buffer. The mixture was incubated at 37°C for 30 minutes to obtain a final concentration of 0.1 μM Cas12a / crRNA complex.

[0045] The fluorescence reaction and corresponding detection were performed as follows: In a 0.6 mL centrifuge tube, the following reaction components were added sequentially: 35 μL water, 5.0 μL 10×CutSmart buffer, 2.0 μL reporter probe (1.0 μM), 2.0 μL dumbbell probe (0.2 μM), 2.0 μL LSA1 (0.2 μM), 2.0 μL Cas12a / crRNA complex (25 nM), and 2.0 μL of FEN1 enzyme at different concentrations. The total reaction volume was 50 μL. The reaction was incubated at 37 °C, and the real-time fluorescence intensity was recorded throughout the reaction.

[0046] Gel characterization was performed as follows: In a 0.6 mL centrifuge tube, the following reaction components were added sequentially: 35 μL water, 5.0 μL 10×CutSmart buffer, 2.0 μL reporter probe (1.0 μM), 2.0 μL dumbbell probe (0.2 μM), 2.0 μL SA1 (0.2 μM), 2.0 μL Cas12a / crRNA complex (25 nM), and 2.0 μL FEN1 enzyme (32 U / μL). The total reaction volume was 50 μL. This 50 μL reaction mixture was incubated at 37 °C for 60 minutes to obtain the sample solution for polyacrylamide gel electrophoresis (PAGE).

[0047] The rationality of the designed sequence was verified by PAGE. The 12% PAGE preparation method was as follows: acrylamide solution (10 mL, 40%), 5×TBE buffer (5.0 mL, 88 mM Tris, 88 mM boric acid, 2.0 mM EDTA, pH=8.3), N,N,N',N'-tetramethylethylenediamine (18 μL), ammonium persulfate (180 μL, 0.1 g / mL), and DEPC water (10 mL). PAGE was performed at 37 °C in constant flow mode (30 mA) for 60 minutes. Finally, the gel was stained in SYBR gold solution in the dark for 40 minutes and photographed using a gel imaging system.

[0048] The technical solution of the present invention will be further explained and described below with reference to specific embodiments.

[0049] Example 1: Feasibility of Detecting FEN1 Activity Using the FCT-CRISPR Strategy In this embodiment, the concentration of the Cas12a / crRNA complex was 25 nM, the concentration of SA1 (SEQ ID NO: 2) was 200 nM, the concentration of SA2 (SEQ ID NO: 3) was 200 nM, the concentration of the dumbbell probe (SEQ ID NO: 1) was 200 nM, the concentration of the reporter probe (SEQ ID NO: 5) was 1600 nM, and the reaction temperature was 37 °C.

[0050] First, the synergistic activation effect of the cleavage activators on the trans-cleavage activity of CRISPR / Cas12a was verified. Two cleavage activators were designed and named SA1 (SEQ ID NO: 2) and SA2 (SEQ ID NO: 3). Figure 2 As shown in Figures A and B, when SA1 is present but SA2 is absent, the fluorescence intensity of the system remains at a low level for 60 minutes, indicating that the trans-cleavage activity is hardly activated under these conditions. Conversely, when both SA1 and SA2 are present, the fluorescence intensity of the system increases significantly within 60 minutes, indicating that the trans-cleavage activity is effectively activated. These results confirm that the cleavage activators (SA1 and SA2) can activate the trans-cleavage activity of CRISPR / Cas12a, further supporting the rational design of the FCT-CRISPR strategy.

[0051] Furthermore, SA2 was replaced with a dumbbell probe (SEQ ID NO: 1) containing a protruding SA2 moiety. Real-time changes in system fluorescence intensity before and after FEN1 treatment were observed. Without FEN1, no increase in system fluorescence intensity was observed. This indicates that although the dumbbell probe contains SA2, it failed to activate trans-cleavage activity. This is likely due to the significant steric hindrance of the dumbbell probe. With the presence of FEN1, the system fluorescence intensity gradually increased over time. This indicates that trans-cleavage activity was activated. This phenomenon may be because FEN1 cleaves the dumbbell probe, causing SA2 dissociation and reducing steric hindrance. These results preliminarily confirm the feasibility of the FCT-CRISPR strategy.

[0052] To further verify feasibility, we investigated the gel electrophoresis behavior of the probe under different conditions. For example... Figure 2 As shown in lane C, lane M represents the DNA marker. Lanes 1 to 5 correspond to the following samples: dumbbell probe (lane 1), dumbbell probe + FEN1 (lane 2), reporter probe (lane 3), dumbbell probe + SA1 + reporter probe + Cas12a / crRNA (lane 4), and dumbbell probe + SA1 + reporter probe + Cas12a / crRNA + FEN1 (lane 5). No dumbbell probe band was observed in lane 2, but two new bands with faster migration rates appeared, indicating that the dumbbell probe could be recognized and cleaved by FEN1, forming two cleavage products. The bands in lane 4 correspond to the positions of the dumbbell probe and reporter probe bands, indicating that in the absence of FEN1, the dumbbell probe remained intact, and the reporter probe was not cleaved. In the presence of FEN1 (lane 5), no bands were observed at the positions corresponding to the dumbbell probe and reporter probe, indicating that both the dumbbell probe and reporter probe were degraded. This may be because the product of FEN1 cleavage of the dumbbell probe activated the trans-cleavage activity of Cas12a, thereby cleaving the reporter probe. These results are consistent with expectations and further confirm the feasibility of the proposed strategy.

[0053] To verify whether the proposed method can achieve read-on-mix operation, real-time fluorescence monitoring was performed. FEN1 to be detected was added to the monitoring reaction system 10 minutes later, and the change in fluorescence intensity was observed—the results are as follows: Figure 2 Figures D and E are shown in the diagram. The purple and blue curves represent the systems containing FEN1 and those without, respectively. FEN1 was added to the positive system at the time points indicated by the arrows. Before the addition of FEN1, the fluorescence intensity of both the positive and negative systems remained at low levels, consistent with previous results. After the addition of FEN1, the fluorescence signal of the positive system increased significantly over time. These results further confirm that the generation of the fluorescence signal is a result of the action of FEN1, and also demonstrate the excellent mixing and readability performance of the proposed method.

[0054] Example 2: The impact of key parameters on the FCT-CRISPR strategy The performance of the FCT-CRISPR strategy is affected by several key parameters, including the number of hybridization bases of SA1 / SA2 with crRNA, the number of spacer bases between SA1 and SA2, the length of the 3'-end flap of the dumbbell probe, the size of the dumbbell probe, and the binding site of the dumbbell probe to crRNA.

[0055] (1) Effect of reaction activator on the number of bases in crRNA hybridization: Reaction system and conditions: The concentration of the Cas12a / crRNA complex was 25 nM, the concentration of SA1 (the sequences with complementary bases of 8, 9, and 10 nt are SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively) was 200 nM, the concentration of SA2 (the sequences with complementary bases of 8, 9, and 10 nt are SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively) was 200 nM, the concentration of the dumbbell probe (SEQ ID NO: 1) was 200 nM, the concentration of the reporter probe (SEQ ID NO: 5) was 1600 nM, and the reaction temperature was 37 °C.

[0056] Hybridization of the activator with crRNA is a prerequisite for activating trans-cleavage activity, and the number of hybridization bases significantly affects the sensitivity and selectivity of the method. This example investigated the changes in fluorescence intensity when the complementary base numbers of SA1 / SA2 and crRNA were 8, 9, and 10, respectively. The results are as follows: Figure 3 As shown in Figures A to C, when the number of bases is 8, the fluorescence signal in the positive control system is at a low level, presumably because insufficient bases make it difficult for SA to bind stably to crRNA. When the number of bases is 10, the negative signal increases significantly, possibly because excessive bases cause a single SA fragment to partially activate trans-cleavage activity. When the number of bases is 9, the negative signal intensity is low, and the positive control signal is significantly stronger than the negative control signal. This indicates that when both SA1 and SA2 have 9 complementary bases, this strategy can not only effectively control the negative signal but also efficiently activate trans-cleavage activity.

[0057] (2) The effect of the spacer region between the two SAs on the trans-cutting activity: Reaction system and conditions: The concentration of Cas12a / crRNA complex was 25 nM, the concentration of SA1 (SEQ ID NO: 2) was 200 nM, the concentration of SA2 (sequences of spacer bases 0, 1, and 2 nt were SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively) was 200 nM, the concentration of dumbbell probe (SEQ ID NO: 1) was 200 nM, the concentration of reporter probe (SEQ ID NO: 5) was 1600 nM, and the reaction temperature was 37 °C.

[0058] To investigate whether the spacer region between two SAs affects trans-cleavage activity, the fluorescence intensity of the positive control system was measured when there were 0, 1, and 2 spacer bases between the two SAs. The results are as follows: Figure 3 As shown in D to F. When the number of spacers is 0, the positive control system outputs a strong fluorescence signal. When there are 1 or 2 spacer bases, there is almost no fluorescence output, indicating that the presence of spacers between SAs prevents the activation of trans-cleavage activity. Therefore, two SAs must be directly adjacent (without spacers) to ensure normal activation of Cas12a trans-cleavage activity.

[0059] (3) Investigation on the effect of the length of the 3'-end flap of the dumbbell probe on the activity of FEN1 endonuclease: Reaction system and conditions: The concentration of Cas12a / crRNA complex was 25 nM, the concentration of SA1 (SEQ ID NO: 2) was 200 nM, the concentration of SA2 (SEQ ID NO: 3) was 200 nM, the concentration of dumbbell probe (the sequences of the 3'-end flap length -1, 0, 1, and 5 nt were SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively) was 200 nM, the concentration of reporter probe (SEQ ID NO: 5) was 1600 nM, and the reaction temperature was 37℃.

[0060] The length of the 3' end flap of the dumbbell probe is a factor affecting the activity of the FEN1 endonuclease. The fluorescence intensity of the positive system changes when the length of the 3' end flap of the dumbbell probe is -1, 0, 1, and 5 nt, respectively. Figure 3 As shown in G to I. When the 3'-end flap length is 0, 1, and 5 nt, the positive signal is strong and the difference is small. When the length is -1 nt, the positive signal is significantly weakened, indicating that an excessively short 3'-end flap length will inhibit FEN1 from cutting the substrate.

[0061] (4) The effect of dumbbell probe size on the control of negative signals: Reaction system and conditions: The concentration of Cas12a / crRNA complex was 25 nM, the concentration of SA1 (SEQ ID NO: 2) was 200 nM, the concentration of SA2 (SEQ ID NO: 3) was 200 nM, the concentration of dumbbell probes (probe sequences with stem lengths of 6, 12, and 18 were SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively) was 200 nM, the concentration of reporter probe (SEQ ID NO: 5) was 1600 nM, and the reaction temperature was 37℃.

[0062] In this study, the spatial steric hindrance effect of the dumbbell probe is the key mechanism for controlling the negative signal, and the size of the dumbbell probe is a key parameter affecting the strategy's operation. Dumbbell probes of different stem lengths (6, 12, and 18 nt) control the negative signal, such as... Figure 3 As shown in J to L, dumbbell probes with stem lengths of 6, 12, and 18 nt all exhibited good negative control.

[0063] (5) The effect of the relative positions of the dumbbell probe and Cas12a enzyme on the negative control effect: Reaction system and conditions: The concentration of Cas12a / crRNA complex was 25 nM, the concentration of SA1 (SEQ ID NO: 2) was 200 nM, the concentration of SA2 (SEQ ID NO: 3) was 200 nM, the concentration of dumbbell probe (the sequences of the internal probe and the external probe are SEQ ID NO: 23 and SEQ ID NO: 24, respectively) was 200 nM, the concentration of reporter probe (SEQ ID NO: 5) was 1600 nM, and the reaction temperature was 37℃.

[0064] The relative position of the dumbbell probe to the Cas12a enzyme may affect the negative control effect. This embodiment designed two dumbbell probe modes—an internal mode and an external mode. In the internal mode, the dumbbell portion is located at the junction of the two cleavage activators, i.e., in the middle position; in the external mode, the dumbbell portion is located on one side of the cleavage activator, i.e., at the edge position. The results are as follows: Figure 3 As shown in M, this indicates that the control effect of the negative signal is better in the internal mode than in the external mode, that is, the spatial steric hindrance effect of the dumbbell probe is more significant in the internal mode.

[0065] (6) Report probe type research: Reaction system and conditions: The concentration of Cas12a / crRNA complex was 25 nM, the concentration of SA1 (SEQ ID NO: 2) was 200 nM, the concentration of SA2 (SEQ ID NO: 3) was 200 nM, the concentration of dumbbell probe (SEQ ID NO: 1) was 200 nM, the concentration of reporter probe (linear structure and hairpin structure sequences were SEQ ID NO: 5 and SEQ ID NO: 6, respectively) was 1600 nM, and the reaction temperature was 37℃.

[0066] The results are as follows Figure 4 As shown, the two reporter probes produce similar signal intensities, but the linear reporter probe exhibits a faster kinetic curve. This difference may be due to the slower separation rate of the fluorophore from the quencher caused by stem hybridization in the hairpin reporter probe.

[0067] Example 3 Optimization of Reaction Conditions In this embodiment, the SA1 sequence is SEQ ID NO: 2, the SA2 sequence is SEQ ID NO: 3, the dumbbell probe sequence is SEQ ID NO: 1, and the report probe sequence is SEQ ID NO: 5.

[0068] (1) Cas12a concentration optimization. Cas12a concentrations were set to 10 nM, 25 nM, 50 nM, 100 nM, and 200 nM, respectively. SA1 concentration was 200 nM, SA2 concentration was 200 nM, dumbbell probe concentration was 200 nM, reporter probe concentration was 1600 nM, and reaction temperature was 37℃. Figure 5 As shown in Figures A and B, the ratio of fluorescence intensity between the negative and positive systems under different Cas12a concentrations shows a trend of increasing and then decreasing. With increasing Cas12a concentration, the negative signal increases significantly, indicating non-specific trans cleavage under high Cas12a concentrations. The ratio of positive to negative signal is highest when the Cas12a concentration is 25 nM; therefore, 25 nM is selected as the optimal Cas12a concentration parameter.

[0069] (2) Optimization of dumbbell probe concentration. The dumbbell probe concentrations were set to 40 nM, 80 nM, 200 nM, 400 nM, and 2000 nM, respectively. The Cas12a concentration was 25 nM, the SA1 concentration was 200 nM, the SA2 concentration was 200 nM, the reporter probe concentration was 1600 nM, and the reaction temperature was 37℃. Figure 6As shown in Figures A and B, the dumbbell probe contains a partial activator of Cas12a. Sufficient dumbbell probe is needed to ensure Cas12a activation, while non-specific Cas12a activation due to excessive dumbbell probe needs to be controlled. The results show that as the dumbbell probe concentration increases, the positive signal increases significantly, while the negative signal increases slightly. The ratio of positive to negative signal is highest when the dumbbell probe concentration is 200 nM. At this concentration, Cas12a is effectively activated and the negative signal is well controlled; therefore, 200 nM is selected as the optimal dumbbell probe concentration.

[0070] (4) Optimization of reporter probe concentration. The reporter probe concentrations were set to 80 nM, 400 nM, 800 nM, 1600 nM, and 40000 nM, respectively. The Cas12a concentration was 25 nM, SA1 concentration was 200 nM, SA2 concentration was 200 nM, the dumbbell probe concentration was 200 nM, and the reaction temperature was 37°C. The results are as follows: Figure 7 As shown in Figures A and B, insufficient reporter probes result in low positive signals, while excessive reporter probes lead to high negative signals due to the accumulation of autofluorescence. Therefore, optimization is necessary. Results indicate that a reporter probe concentration of 1600 nM results in significant positive signal intensity and good control of negative signals, with the highest positive to negative signal ratio. Therefore, 1600 nM is determined to be the optimal reporter probe concentration.

[0071] Example 4: Analytical performance of the FCT-CRISPR strategy for detecting FEN1 activity After systematic parameter optimization, this embodiment evaluates the performance of the constructed analytical method. This embodiment uses the reaction system optimized in Example 3, with SA1 sequence SEQ ID NO: 2, SA2 sequence SEQ ID NO: 3, dumbbell probe sequence SEQ ID NO: 1, and reporter probe sequence SEQ ID NO: 5.

[0072] First, the fluorescence intensity of the system was measured in the presence of different concentrations of FEN1 enzyme. For example... Figure 8 As shown in Figure A, the fluorescence signal intensity gradually increases with increasing FEN1 enzyme concentration. When the FEN1 enzyme concentration is 6.4 × 10⁻⁶, the fluorescence signal intensity increases. -1 At U, the signal intensity reaches its peak. Based on the above data, a concentration-fluorescence intensity relationship graph is generated, as shown below. Figure 8 As shown in B and C. At 6.4 × 10 -4 ~6.4×10 -1Within the U range, FEN1 concentration and fluorescence signal intensity exhibited a good linear relationship, with the linear equation F = 14417C_FEN1 + 180.11 and a correlation coefficient (R²) of 0.993. According to the 3σ rule, the limit of detection (LOD) of this method was calculated using the formula LOD = 3SD / k, where SD is the standard deviation of the blank sample signal value, and k represents the slope of the calibration curve. The LOD of this method was calculated to be 0.2 mU. Compared with previously reported methods, the proposed method exhibits satisfactory sensitivity.

[0073] To evaluate the selectivity of the FCT-CRISPR strategy, the signal intensities of the FEN1 enzyme and different interfering components were recorded. Five interfering components were investigated, including bovine serum albumin (BSA), lysozyme, acetylcholinesterase (AChE), trypsin, and DpnI. Furthermore, the signal intensity of FEN1 samples treated with ATA (Aurintricarboxylic Acid, a validated FEN1 enzyme inhibitor that inhibits FEN1 by interfering with protein-nucleic acid interactions) was also measured. Figure 8 As shown in Figures D and E, the signal intensity of the system in the presence of interfering components is similar to that of the blank control, remaining at a low level. When the FEN1 enzyme is present, the system outputs a strong fluorescence signal. Conversely, when the FEN1 enzyme is inhibited by ATA, the fluorescence signal of the system significantly decreases. These results confirm the excellent selectivity of this method.

[0074] Example 5: Application in actual biological sample detection In this embodiment, the reaction system optimized in Example 3 is used. The SA1 sequence is SEQ ID NO: 2, the SA2 sequence is SEQ ID NO: 3, the dumbbell probe sequence is SEQ ID NO: 1, and the reporter probe sequence is SEQ ID NO: 5.

[0075] FEN1 is typically aberrantly overexpressed in cancer cells. Therefore, we used MCF-7 and HeLa cells as models to evaluate the ability of the proposed method to detect real biological samples. Figure 9 Figure A illustrates the proposed method for detecting biological samples. First, proteins were extracted from MCF-7 and HeLa cells to obtain biological samples containing the FEN1 enzyme. Then, the obtained biological samples were added to the detection system, and after mixing and reaction, the fluorescence signal intensity was read. Extracts from MCF-7 and HeLa cells, as well as their heat-treated extracts, were detected. Results are shown below. Figure 9As shown in B and C, the fluorescence intensity of MCF-7 and HeLa cell extracts was significantly stronger than that of the blank control. Conversely, the signal intensity of the heat-treated samples was similar to that of the blank control, which may be due to the inactivation of the FEN1 enzyme caused by heating. Furthermore, we preliminarily measured the fluorescence intensity of different numbers of HeLa cells (50, 200, 500, 800, and 1000 cells). The results are as follows... Figure 10 As shown, fluorescence intensity increases with increasing cell number, exhibiting a good linear relationship in the range of 50–1000 cells. This method can detect cells as low as 20, demonstrating good sensitivity. These results indicate that the proposed method is suitable for the detection of real-world biological samples.

[0076] In summary, this study developed a DNA flap-controlled CRISPR / Cas12a trans-cleavage (FCT-CRISPR) strategy with a direct activation mechanism. The FEN1 enzyme specifically cleaves the dumbbell probe, eliminating steric hindrance that prevents the activator fragment from binding to the Cas12a protein. Then, the trans-cleavage activity is activated synergistically with another activator fragment. This trans-cleavage activity hydrolyzes multiple molecular beacons, releasing an amplified fluorescent signal, thus providing excellent sensitivity. The entire sensing process requires only a single mixing operation to read the signal, eliminating the need for additional enzyme tools or separation steps. This mix-and-read approach reduces errors introduced by multi-step operations and is easy to implement. The proposed strategy can detect FEN1 enzyme levels as low as 0.2 mU, exhibiting good sensitivity. Selectivity experiments demonstrate the strategy's excellent anti-interference capability. It also demonstrated good analytical performance in practical biological sample detection, successfully achieving the analysis of FEN1 activity in cancer cells. This provides sensitive, accurate, and convenient sensing technology support for FEN1 enzyme activity detection, and has considerable application potential in early disease diagnosis and basic biomedical research.

[0077] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A DNA flap controlled FCT-CRISPR detection system, characterized in that, The dumbbell probe comprises a DNA flap structure, a CRISPR / Cas12a protein, a crRNA, a reporter probe and a Cas12a split activator SA1. The DNA flap structure comprises a 5'-end flap and a 3'-end flap, the 5'-end flap is a Cas12a split activator SA2, and the 5'-end flap can be cut by a flap endonuclease 1.

2. The FCT-CRISPR detection system of claim 1, wherein, The stem length of the dumbbell probe is 6-18 nt.

3. The FCT-CRISPR detection system of claim 1, wherein, The 5' end of the reporter probe is modified with a fluorescent group, and the 3' end is modified with a quenching group.

4. The FCT-CRISPR detection system of claim 1, wherein, SA1 and SA2 have the same number of complementary bases with the crRNA, and the number of bases is 8-9 nt.

5. The FCT-CRISPR detection system of claim 1, wherein, The reporter probe has a linear structure or a hairpin structure.

6. The FCT-CRISPR detection system of claim 1, wherein, It further comprises a buffer.

7. A test kit comprising, The FCT-CRISPR detection system of any one of claims 1-6.

8. Use of the FCT-CRISPR detection system of any one of claims 1-6 or the detection kit of claim 7 in detecting the activity of a flap endonuclease 1, which is not for the purpose of diagnosis and treatment of diseases.

9. Use according to claim 8, wherein the compound is ###0002### It comprises the following steps: S1, incubating CRISPR / Cas12a protein and crRNA in a buffer to obtain a Cas12a / crRNA complex; S2, mixing the dumbbell probe, Cas12a split activator SA1, Cas12a / crRNA complex, reporter probe and buffer to obtain a reaction system; S3, adding the sample of the flap endonuclease 1 to be tested into the reaction system, performing constant temperature reaction, detecting the generated fluorescence signal and analyzing the results.

10. Use according to claim 9, characterized in that, The working concentration range of the dumbbell probe is 0.1-0.3 μM, the working concentration range of the Cas12a split activator SA1 is 0.1-0.3 μM, the working concentration range of the Cas12a / crRNA complex is 20-30 nM, the working concentration range of the reporter probe is 0.8-1.2 μM, and the constant temperature reaction condition is 25-37℃ for 20-70 min.

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