Stimulus-responsive CRISPR-Cas12a system and application thereof

Abstract

The invention relates to a stimulus-responsive CRISPR-Cas12a (clustered regularly interspaced short palindromic repeats-associated 12a) system and application thereof, and belongs to the technical field of biology. The CRISPR-Cas12a (clustered regularly interspaced short palindromic repeats-CRISPR-Cas12a) system with the stimulation response comprises a DNA (deoxyribonucleic acid) tetrahedron-stimulation response factor aptamer compound and a CRISPR-Cas12a reaction system, the DNA tetrahedron-stimulus response factor aptamer compound is formed by hybridizing a DNA tetrahedron and a stimulus response factor aptamer; the DNA tetrahedron is formed by self-assembly of TH1, TH2, TH3 and a target chain TS of a CRISPR-Cas12a reaction system, and the stimuli response factor aptamer and the TS are hybridized so that the stimuli response factor and the stimuli response factor aptamer are combined to expose the TS under the action of the stimuli response factor, and then the CRISPR-Cas12a reaction system is activated. The H1 / H3 treated by the CRISPR-Cas12a with stimulation response can effectively inhibit the phosphorylation level of c-Met and the phosphorylation level of downstream protein of the c-Met, and inhibit the proliferation, migration and invasion ability of cells. The invention realizes specific control of membrane receptor dimerization and corresponding cell behaviors in a customizable manner.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a stimulus-responsive CRISPR-Cas12a system and its applications. Background Technology

[0002] Lung cancer is the most common cancer worldwide and a leading cause of cancer death. Studies have found that lung cancer has consistently high incidence and mortality rates globally. Lung cancer patients are classified by tissue subtype into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), with NSCLC accounting for approximately 85% of lung cancer cases. The c-Met gene, as a proto-oncogene, is closely related to tumor development, progression, prognosis, and outcome due to overactivation. Research indicates that c-Met receptor dimerization activation participates in the formation and development of NSCLC. Abnormal activation of c-Met (such as exon 14 skipping mutations, gene amplification, and protein overexpression) leads to increased receptor dimerization, which in turn continuously activates downstream signaling pathways, promoting the growth, invasion, and metastasis of lung cancer cells. Blocking the dimerization process caused by ligand binding to c-Met can effectively inhibit the growth, invasion, and metastasis of lung cancer cells, providing a new approach to cancer treatment. Furthermore, by detecting the c-Met gene status and protein expression levels, doctors can develop corresponding treatment plans based on the patient's condition, promoting the development of personalized medicine. Therefore, developing new and effective strategies targeting the c-Met receptor to modulate its receptor function is of great significance for targeted therapy of non-small cell lung cancer.

[0003] Cell membrane receptors are important protein structures on the cell surface, capable of sensing and transmitting signals from extracellular chemical or physical stimuli, thereby regulating cellular physiological and pathological processes. Common membrane receptors include receptor tyrosine kinases (RTKs), T cell receptors (TCRs), and G protein-coupled receptors (GPCRs), which play crucial roles in cell proliferation, differentiation, apoptosis, metabolism, and migration. Typically, receptor oligomerization plays a vital role in activating downstream intracellular signaling cascades to control cellular responses to extracellular signals. c-Met is a typical RTK; after its extracellular domain binds to hepatocyte growth factor (HGF), the c-Met receptor dimerizes, activating its intracellular tyrosine kinase activity, further activating downstream signaling pathways such as PI3K-Akt, Ras-MAPK, and STAT, thereby promoting cell proliferation, growth, migration, invasion, and angiogenesis.

[0004] Traditional genetic engineering techniques have been attempted to regulate receptor oligomerization; however, these genetic methods are limited by unpredictability, inefficiency, and high cost. In contrast, DNA nanostructures, as a commonly used non-genetic tool, have been used to regulate receptor function due to their unique advantages of precise base pairing, programmable structure, and flexible modification. For example, long-term monitoring of single-molecule behavior during receptor dimerization using a plasma scale can deepen our understanding of the dimerization process and track the behavior of individual receptors, thereby enabling precise regulation and dynamic monitoring of the dimerization process. Furthermore, DNA nanodevices can also be selectively activated to regulate membrane receptor dimerization. For instance, external light stimulation or the addition of trigger DNA strands can fold hairpin structures in DNA nanoscaffolds, causing their spring-like motion, which can be used to manipulate receptor aggregation. Sethi et al. designed a light-switchable DNA nanostructure containing azophenyl groups, which modulates receptor function by changing its conformation through light irradiation, thereby dynamically adjusting the spatial distance between molecules. However, its selectivity needs further improvement. Vascular endothelial growth factor (VEGF) is a signaling protein secreted by cells that promotes angiogenesis. Because its expression is relatively higher around cancer cells than in normal cells, it is often used to distinguish between different cell types. It is also widely studied as a cancer biomarker and a target for tumor therapy. This makes VEGF a suitable candidate for triggering selective membrane receptor aggregation in cancer cells.

[0005] Furthermore, CRISPR-Cas12a is a nuclease from the type V CRISPR system, possessing both cis and trans-cleavage activities. This enzyme not only exhibits excellent performance in gene editing but also shows great potential for sensitive detection of molecular targets. Its outstanding target recognition and substrate cleavage capabilities make it an ideal signal amplification unit, and its combination with a range of tools (such as nanomaterials and functional nucleic acids) enables specific target detection. For example, the RNA-activated CRISPR-Cas12a nanomachines developed by Yuan et al. utilize the high local concentration effect on the surface of gold nanospheres to significantly enhance the cleavage rate and signal intensity of Cas12a, achieving efficient imaging of miRNAs in living cells. In addition, Cas12a can also achieve rapid and sensitive cancer cell recognition on living cell membranes. For instance, Yao et al. designed a novel detection tool called FINDER, combining CRISPR technology with a fluid-confined DNA reporter gene, enabling efficient recognition of cancer cells in complex physiological environments. Therefore, developing a highly selective system capable of modulating cell membrane receptor function is of great significance for targeted therapy of non-small cell lung cancer. Summary of the Invention

[0006] The first objective of this invention is to provide a stimulus-responsive CRISPR-Cas12a system that enables customized and specific control of membrane receptor dimerization and its corresponding cellular behavior by editing strategies for protein-protein interactions.

[0007] A second objective of this invention is to provide an application of a stimulus-responsive CRISPR-Cas12a system.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] A stimulus-responsive CRISPR-Cas12a system includes a DNA tetrahedron-stimulus response factor aptamer complex and a CRISPR-Cas12a reaction system. The DNA tetrahedron-stimulus response factor aptamer complex is formed by hybridization of a DNA tetrahedron and a stimulus response factor aptamer. The DNA tetrahedron is formed by the self-assembly of TH1, TH2, TH3 and the target strand TS of the CRISPR-Cas12a reaction system. The stimulus response factor aptamer hybridizes with the TS so that, under the action of a stimulus response factor, the stimulus response factor binds to the stimulus response factor aptamer to expose the TS, thereby activating the CRISPR-Cas12a reaction system.

[0010] Furthermore, the nucleotide sequence of TH1 is shown in SEQ ID NO.1; the nucleotide sequence of TH2 is shown in SEQ ID NO.2; the nucleotide sequence of TH3 is shown in SEQ ID NO.3; the nucleotide sequence of TS is shown in SEQ ID NO.4; and the nucleotide sequence of the stimulus response factor aptamer is shown in SEQ ID NO.5.

[0011] Furthermore, the stimulus response factor is VEGF.

[0012] Furthermore, the Tm of the TS is greater than 40°C, and the Tm of the stimulus response factor aptamer is greater than 40°C and less than 50°C after the TS and the stimulus response factor aptamer are hybridized.

[0013] Furthermore, the CRISPR-Cas12a reaction system includes Cas12a protein, crRNA, and NEBuffer r2.1 buffer. The final concentration of Cas12a protein is 25 nmol / L, the final concentration of crRNA is 25 nmol / L, and the molar ratio of crRNA to Cas12a protein is 1:1.

[0014] Furthermore, the molar ratio of TH1, TH2, TH3, and TS is 1:1:1:1; the molar ratio of the DNA tetrahedron to the stimulus-response aptamer is 1:1.

[0015] Application of the stimulus-responsive CRISPR-Cas12a system in regulating cell membrane receptor dimerization.

[0016] Furthermore, the regulation of cell membrane receptor dimerization is the regulation of c-Met receptor dimerization or c-Met receptor dimerization with TfR receptor dimerization.

[0017] Further, the steps for regulating the dimerization of c-Met receptor and TfR receptor are as follows: A549 cells are incubated with the DNA tetrahedron-stimulation response factor aptamer complex, followed by the addition of a stimulation response factor, then a mixed probe of H1 and H3 is added and incubated on ice. After centrifugation, the supernatant is removed to obtain a cell pellet. The cell pellet is then mixed with the CRISPR-Cas12a reaction system and incubated at 37°C for 30 min. The steps for regulating the dimerization of c-Met receptor and c-Met receptor are as follows: A549 cells are incubated with the DNA tetrahedron-stimulation response factor aptamer complex, followed by the addition of a stimulation response factor, then a mixed probe of H1 and H2 is added and incubated on ice. After centrifugation, the supernatant is removed to obtain a cell pellet. The cell pellet is then mixed with the CRISPR-Cas12a reaction system and incubated at 37°C for 30 min. H1 and H2 are nucleic acid aptamer sequences targeting the c-Met receptor, and H3 is a nucleic acid aptamer sequence targeting the TfR receptor. The nucleotide sequence of H1 is as shown in SEQ ID. As described in NO.5, the nucleotide sequence of H2 is as described in SEQ ID NO.6, and the nucleotide sequence of H3 is as described in SEQ ID NO.7.

[0018] The beneficial effects of this invention are:

[0019] c-Met dimerization activation plays a crucial role in the formation and development of NSCLC. Abnormal c-Met activation leads to increased receptor dimerization, which in turn continuously activates downstream signaling pathways, thereby promoting the growth, invasion, and metastasis of lung cancer cells. Blocking the c-Met receptor dimerization process can effectively inhibit the growth and metastasis of lung cancer cells, providing a new approach for targeted therapy of NSCLC. The stimulation-responsive CRISPR-Cas12a-treated H1 / H3 of this invention can effectively inhibit the phosphorylation levels of c-Met and its downstream proteins, and further suppress cell proliferation, migration, and invasion.

[0020] This invention anchors a complex (Td-apt) formed by assembling a DNA tetrahedron and a VEGF aptamer onto the cell membrane. When the stimulus-response factor VEGF is present, VEGF binds to the aptamer, exposing the CRISPR-Cas12a target strand TS, thus activating CRISPR-Cas12a. Then, utilizing the trans-cleavage enzyme activity of CRISPR-Cas12a, the hairpin structures of H1, H2, and H3 are continuously cleaved, promoting the dimerization of c-Met receptors and TfR receptors, thereby inhibiting receptor function, or causing c-Met receptors to dimerize themselves, thereby activating receptor function. By editing protein-protein interactions through the CRISPR-Cas system, this invention achieves customized and specific control over membrane receptor dimerization and its corresponding cellular behavior. Attached Figure Description

[0021] Figure 1 A schematic diagram illustrating the principle of the CRISPR-Cas12a system, which is a stimulus-responsive system, for regulating the selective aggregation of cell membrane receptors.

[0022] Figure 2 Figure 1 shows the electrophoresis, particle size, and Zeta potential characterization results of Td and Td-apt. In Figure 2(a), the electrophoresis results are shown, with lane 1: marker; lane 2: TH1; lane 3: TH2; lane 4: TH3; lane 5: TS; lane 6: VEGF apt (5 μmol / L); lane 7: TH1+TH2; lane 8: TH1+TH2+TH3; lane 9: TH1+TH2+TH3+TS; lane 10: TH1+TH2+TH3+TS+VEGF apt. Figure 2(b) shows the hydrated particle size of Td; Figure 3(c) shows the hydrated particle size of Td-apt; and Figure 4(d) shows the Zeta potential characterization results of Td and Td-apt.

[0023] Figure 3 Flow cytometry analysis of Td-apt stability at the cell membrane level;

[0024] Figure 4 Flow cytometry plot of Td-apt response to VEGF;

[0025] Figure 5 The graph shows the changes in fluorescence intensity when Td-apt is treated with different concentrations of VEGF.

[0026] Figure 6 The images show the fluorescence spectra of CRISPR-Cas12a trans-cleavage activity, where a is the fluorescence spectrum characterization of CRISPR-Cas12a trans-cleavage of H1, b is the fluorescence spectrum characterization of CRISPR-Cas12a trans-cleavage of H2, and c is the fluorescence spectrum characterization of CRISPR-Cas12a trans-cleavage of H3.

[0027] Figure 7 The fluorescence spectra of the Td-apt activated CRISPR-Cas12a reaction system regulating the selective aggregation of cell membrane receptors are shown in Figure a. Figure a is the fluorescence spectrum characterization of H1 / H2 treated with CRISPR-Cas12a in response to stimulation, which regulates the self-dimerization of c-Met receptor. Figure b is the fluorescence spectrum characterization of H1 / H3 treated with CRISPR-Cas12a in response to stimulation, which regulates the dimerization of c-Met / TfR receptor.

[0028] Figure 8 Figure 1 shows the expression levels of c-Met protein in A549 cells and BEAS-2B cells.

[0029] Figure 9 Figure showing the regulatory effect of the Td-apt activated CRISPR-Cas12a reaction system on the phosphorylation level of related proteins in A549 cells;

[0030] Figure 10 Figure showing the regulatory effect of the Td-apt activated CRISPR-Cas12a reaction system on the phosphorylation level of phase-related proteins in BEAS-2B cells;

[0031] Figure 11 The diagram shows the regulatory effect of the Td-apt activated CRISPR-Cas12a reaction system on the migration and invasion abilities of A549 cells, where a represents the regulatory effect on the migration ability of A549 cells and b represents the regulatory effect on the invasion ability of A549 cells.

[0032] Figure 12 Figures show the optimization results of the CRISPR-Cas12a reaction system, where a represents the optimized Cas12a concentration, b represents the optimized crRNA / Cas12a ratio, c represents the optimized buffer solution, d represents the optimized reaction time, and e represents the optimized reaction temperature. Detailed Implementation

[0033] The present invention will be further described below with reference to the embodiments and accompanying drawings.

[0034] Preparation of Hybridization Buffer 1: Weigh 2.4228 g of Tris, 0.9521 g of MgCl2 and 5.8440 g of NaCl using an analytical balance. Dissolve them in 950 mL of autoclaved water in a beaker using a magnetic stirrer. Adjust the pH to 7.4 using a pH meter. Then transfer the solution to a 1000 mL volumetric flask and make up to volume. Invert the flask to mix thoroughly. The final solution contains 20 mmol / L Tris, 10 mmol / L MgCl2 and 0.1 mol / L NaCl.

[0035] Preparation of binding buffer 2: Weigh 0.1000g of BSA, 0.0476g of MgCl2 and 0.5500g of glucose using an analytical balance. Dissolve them in 50mL of 1×PBS in a beaker using a magnetic stirrer. Then add PBS to make up to 100mL and mix the solution evenly by shaking up and down. The final solution is binding buffer 2 containing 1g / L BSA, 5mmol / L MgCl2 and 25mmol / L glucose.

[0036] Example 1

[0037] I. Design of DNA tetrahedral-VEGF aptamer complex

[0038] During the design process, the melting temperature (Tm) of the nucleic acid sequence must meet the following conditions: ① The Tm value of TS is higher than 40℃, and the Tm value of VEGF aptamer (VEGF apt) is higher than 40℃; ② The Tm value of the chain after hybridization of TS and VEGF apt is greater than 40℃ but less than 50℃, to ensure that VEGF can effectively compete for and bind to VEGF apt; ③ In the structure formed by DNA tetrahedron (Td), the sticky ends of the TS chain need to be exposed in order to bind to VEGF apt. The designed nucleic acid sequences are shown in Table 1.

[0039] Table 1 Nucleic Acid Names and Sequences

[0040]

[0041]

[0042] Note: Lowercase "aucg" represents RNA bases; uppercase "ATCG" represents DNA bases.

[0043] II. Construction of a stimulus-responsive CRISPR-Cas12a system

[0044] 1. Preparation method of DNA tetrahedron-VEGF aptamer complex: TH1, TH2, TH3, and TS were mixed in a molar ratio of 1:1:1:1 and reacted in a 95℃ water bath for 5 min. Then, the mixture was co-incubated in a 25℃ constant temperature metal shaker at 500 r / min for 1 h to synthesize DNA tetrahedrons (Td). An equimolar amount of VEGF aptamer was added, and the mixture was reacted in a constant temperature metal bath at 37℃ and 500 r / min for 1 h to obtain DNA tetrahedron-VEGF aptamer complex (Td-apt). The concentrations of the prepared Td and Td-apt were adjusted to 1 μmol / L using hybridization buffer 1.

[0045] 2. Preparation method of CRISPR-Cas12a reaction system: Cas12a protein with a final concentration of 25 nmol / L, crRNA with a final concentration of 25 nmol / L, and NEB buffer r2.1 buffer were mixed in a sterile water vortex and reacted at 37℃ for 15 min.

[0046] The experimental principle of using the stimulus-responsive CRISPR-Cas12a system to regulate the selective aggregation of cell membrane receptors is as follows: Figure 1 As shown, the complex formed by assembling a DNA tetrahedron and a VEGF aptamer is first anchored to the cell membrane. When the stimulus-response factor VEGF is present, VEGF binds to the aptamer, exposing the CRISPR-Cas12a target TS strand and activating CRISPR-Cas12a. Utilizing the trans-cleavage enzyme activity of CRISPR-Cas12a, the hairpin structures of probes H1 and H3 are continuously cleaved, causing H1 and H3 to hybridize according to the base complementarity principle, promoting the dimerization of the c-Met receptor and the TfR receptor, thereby inhibiting receptor function. Alternatively, the hairpin structures of probes H1 and H2 are continuously cleaved, causing H1 and H2 to hybridize according to the base complementarity principle, leading to the dimerization of the c-Met receptor itself, thereby activating receptor function. By utilizing the CRISPR system to edit protein-protein interactions, a customizable and specific control over membrane receptor dimerization and its corresponding cellular behavior can be achieved. In this way, the CRISPR-Cas12a reaction system can regulate the activation or inhibition of c-Met receptor dimerization, as well as the intracellular tyrosine kinase phosphorylation level and downstream signaling pathway activation level. Ultimately, this effectively controls the proliferation, migration, and invasion of membrane receptors. H1 and H2 are nucleic acid aptamer sequences targeting the c-Met receptor, and H3 is a nucleic acid aptamer sequence targeting the TfR receptor.

[0047] from Figure 2As can be seen, the lanes for TH1, TH2, TH3, TS, and VEGF apt all show a clear band. This indicates that the TH1+TH2 band is significantly higher than the TH1 or TH2 band alone, suggesting that TH1 and TH2 hybridize through base pairing. Similarly, the TH1+TH2+TH3 band is significantly higher than the TH1+TH2 band, indicating that TH1, TH2, and TH3 hybridize through base pairing. The Td band (TH1+TH2+TH3+TS) is significantly higher than the TH1+TH2+TH3 band, indicating that Td hybridizes through base pairing. Finally, the Td-apt band (TH1+TH2+TH3+TS+apt) is significantly higher than the Td band, indicating that Td-apt hybridizes through base pairing. With the addition of TH1, TH2, TH3, TS, and VEGF apt chains, the molecular weight of the nucleic acid increased, the migration rate slowed down, and the band position gradually shifted upward, indicating that Td and Td-apt were successfully prepared. Dynamic light scattering (DLS) technology determined the particle size of Td to be 11.05 nm and the zeta potential to be -6.45 mV. In contrast, the particle size of Td-apt was 17.66 nm and the zeta potential was -7.19 mV. This shows that Td-apt has a larger particle size and a more negative zeta potential than Td, indicating the successful preparation of both Td and Td-apt.

[0048] III. Stability analysis of Td-apt anchorage on the cell membrane

[0049] 1. Cell preparation: A549 cells in logarithmic growth phase were collected after trypsin digestion, centrifuged and resuspended, and the cell density was adjusted to 2 × 10⁻⁶ cells / year. 5 cell / mL. Transfer the cell suspension to 5mL centrifuge tubes, adding 1mL of cell suspension to each tube.

[0050] 2. Probe Incubation: FAM-Td-apt-BHQ1 was incubated with A549 cells. The A549 cell group served as the negative control group, and the Td-FAM group served as the positive control group. FAM-Td-apt-BHQ1 at different time points served as the experimental group, and changes in cell fluorescence were detected by flow cytometry. Fluorescence in the FAM channel was collected under 488nm excitation, as shown below. Figure 3 As shown, after Td-apt anchors to the cell membrane, its fluorescence remains quenched for 120 minutes, indicating that Td-apt has high stability on the cell membrane surface.

[0051] IV. Characterization of the VEGF response performance of cell membrane-anchored Td-apt

[0052] 1. Cell preparation: The cell preparation method is the same as that used for stability analysis of Td-apt anchoring on the cell membrane.

[0053] 2. Probe Incubation: After centrifuging the cell suspension and removing the supernatant, the cell pellet was mixed thoroughly with Td-FAM, FAM-Td-apt-BHQ1, and binding buffer 2. VEGF was then added, and the mixture was incubated at 37°C with a metal shaker for 30 min. The final concentration of FAM-Td-apt-BHQ1 was 200 nmol / L, and the final reaction volume was 200 μL. Fluorescence intensity was detected using flow cytometry. Figure 4 As shown, fluorescence from the FAM channel was collected under 488 nm excitation. The addition of apt-BHQ1 quenched the fluorescence; however, the addition of VEGF eliminated the quenching, and the fluorescence intensity significantly increased. This indicates that Td-apt can respond to VEGF stimulation, and that Td and Td-apt can bind to the cell membrane via cholesterol. Furthermore, from... Figure 5 It can be seen that when Td-apt is treated with different concentrations of VEGF, the fluorescence intensity gradually increases with the increase of VEGF concentration, indicating that the response of Td-apt to VEGF is concentration-dependent.

[0054] V. Characterization of Td-apt activation of CRISPR-Cas12a trans-cleavage activity

[0055] 1. Cell preparation: The cell preparation method is the same as that used for stability analysis of Td-apt anchoring on the cell membrane.

[0056] 2. Probe Preparation: After incubating Td-apt with A549 cells for 15 min, VEGF was added and reacted for 30 min. Then, 200 nmol / L H1, H2, or H3 was added and incubated on ice. After discarding the solution, the cells were added to the CRISPR-Cas12a reaction system for co-incubation. Real-time imaging of live cells was performed using a confocal microscope. Excitation wavelengths were set to 552 nm (H1), 638 nm (H2), and 638 nm (H3), and the corresponding fluorescence signal ranges were collected. Figure 6 As shown, fluorescence was collected at 560nm-750nm (H1), 660nm-750nm (H2), and 660nm-750nm (H3). Upon addition of stimulus-responsive CRISPR-Cas12a, crRNA binds to the TS strand, activating the trans-cleavage activity of CRISPR-Cas12a, which then cleaves the hairpin structures of H1, H2, and H3, resulting in increased fluorescence intensity. This experimental result demonstrates that the Cas12a protein possesses a strong trans-cleavage capability.

[0057] VI. Characterization of the Td-apt-activated CRISPR-Cas12a response system regulating selective aggregation of cell membrane receptors

[0058] 1. Cell preparation: The cell preparation method is the same as that used for stability analysis of Td-apt anchoring on the cell membrane.

[0059] 2. Probe Preparation: After incubating Td-apt with A549 cells for 15 min, VEGF was added and reacted for 30 min. Then, a mixed probe of H1 and H2 or H1 and H3 was added, and the mixture was incubated on ice for 15 min. The supernatant was removed by centrifugation, and the CRISPR-Cas12a reaction system was added and incubated at 37℃ with a metal shaker at 500 rpm for 30 min. In the presence of CRISPR-Cas12a, crRNA binds to TS, activating the trans-cleavage activity of CRISPR-Cas12a, cleaving the hairpin structures of H1-Cy3-BHQ2, H2-Cy5-BHQ2, and H3-Cy5-BHQ2. The cleaved H1-Cy3-BHQ2 and H2-Cy5-BHQ2, as well as H1-Cy3-BHQ2 and H3-Cy5-BHQ2, due to their close proximity in double-strand hybridization, displace BHQ1, resulting in FRET. Figure 7 As shown, fluorescence in the Cy5 channel (570nm-750nm) was collected under 550nm excitation. The probe without stimulus-responsive CRISPR-Cas12a treatment showed no significant peak at 688nm, while the probe treated with stimulus-responsive CRISPR-Cas12a showed increased fluorescence intensity at 688nm (indicated by the black arrow). This result indicates that the stimulus-responsive Td-apt-activated CRISPR-Cas12a reaction system can regulate the selective aggregation of cell membrane receptors.

[0060] VII. c-Met protein expression levels in A549 and BEAS-2B cells

[0061] 1. Cell Plating: A549 and BEAS-2B cells were collected after trypsin digestion, centrifuged and resuspended, and then plated into culture dishes, adding 2 mL of cell suspension to each well. When the cells reached 80% confluence, the culture medium was discarded, and the cells were washed three times with PBS and then starved for 24 h.

[0062] 2. c-Met protein analysis: The expression level of c-Met in the two cell types was detected by Western blotting. Figure 8 It can be seen that the expression level of c-Met in A549 cells is higher than that in BEAS-2B cells.

[0063] VIII. The regulatory effect of the Td-apt activated CRISPR-Cas12a reaction system on the phosphorylation level of related proteins in A549 cells and BEAS-2B cells.

[0064] A549 cells:

[0065] 1. Cell plating: A549 cells were trypsin-digested, collected, centrifuged, resuspended, and the cell density was adjusted to 1×10⁻⁶. 5 Cells / mL. Seed the cells in a culture dish, adding 2 mL of cell suspension to each well. When the cells reach 80% confluence, discard the culture medium, wash three times with PBS, and then starve the cells for 24 hours.

[0066] 2. Cellular Intervention: Taking H1 / H3 as an example, after incubating Td-apt with cells for 15 min, VEGF was added and the reaction proceeded for 30 min. Then, a mixed probe of H1 and H3 (500 nmol / L) was added and incubated on ice for 15 min. After discarding the solution, the CRISPR-Cas12a reaction system was added and incubated at 37°C for 30 min. The cells were washed three times with PBS, and 30 ng / mL HGF was added, followed by an incubation at 37°C for 30 min. Figure 9 It can be seen that, compared with the HGF group, the expression levels of p-Met, p-Akt, and p-Erk1 / 2 were reduced in the H1 / H3 experimental group treated with CRISPR-Cas12a, while the expression levels of p-Met, p-Akt, and p-Erk1 / 2 remained basically unchanged in the H1 / H3 control group without CRISPR-Cas12a treatment. This indicates that the H1 / H3 treated with the CRISPR-Cas12a reaction system can effectively inhibit the phosphorylation levels of c-Met and downstream proteins.

[0067] BEAS-2B cells: treated in the same way as A549 cells, from... Figure 10 It can be seen that, compared with the HGF-treated group, the expression levels of p-Met, p-Akt, and p-Erk1 / 2 in the H1 / H3 experimental group treated with stimulus-response CRISPR-Cas12a remained essentially unchanged. This result indicates that the stimulus-response Td-apt activated CRISPR-Cas12a response system has good selectivity and can specifically regulate c-Met and its downstream signaling pathways.

[0068] IX. The regulatory effect of the Td-apt activated CRISPR-Cas12a reaction system on cell proliferation.

[0069] 1. Cell plating: A549 cells in logarithmic growth phase were collected after trypsin digestion, centrifuged and resuspended, and the cell density was adjusted to 5 × 10⁶ cells / year. 5 Add 100 μL of the solution per well and continue culturing the cells.

[0070] 2. Probe Intervention: After culturing A549 cells for 18 hours, the cells were washed three times with PBS. The prepared Td-apt was incubated with the cells for 15 minutes, followed by the addition of VEGF and a reaction time of 30 minutes. Taking H1 / H3 as an example, mixed probes of H1 and H3 at concentrations of 0 nmol / L, 10 nmol / L, 30 nmol / L, 50 nmol / L, 100 nmol / L, and 200 nmol / L were added respectively and incubated on ice for 15 minutes. After discarding the solution, the CRISPR-Cas12a reaction system was added, and the cells were incubated at 37°C for 30 minutes. The cells were then washed three times with PBS, and 30 ng / mL HGF was added, followed by a reaction at 37°C for 30 minutes.

[0071] 3. Add CCK-8: Wash three times with PBS, add CCK-8; the culture medium is a 1:19 solution, add 200 μL to each well, incubate for 1 h, and measure the absorbance. The proliferation inhibition rate formula is:

[0072]

[0073] Table 2. Effects of the Td-apt-activated CRISPR-Cas12a response system on cell proliferation (n=3)

[0074]

[0075]

[0076] Note: # indicates P < 0.05, meaning the experimental group was statistically significant compared to the HGF group.

[0077] 10. Regulatory Effects on Cell Migration Ability

[0078] 1. Cell plating: After digesting and centrifuging A549 cells, adjust the cell suspension to 1×10⁻⁶. 5 After adding 1 mL of the solution to each well, continue culturing. When the cells reach 80% confluence, starve them for 24 hours.

[0079] 2. Cell streaking: Using a 200 μL sterile pipette tip, streak a line perpendicular to the bottom of the cell and photograph it under a microscope. This step is recorded as 0h. After incubating the prepared Td-apt with the cells for 15 min, add VEGF and react for 30 min.

[0080] 3. Probe Intervention: Taking H1 / H3 as an example, add H1 / H3 and incubate on ice for 15 min. After discarding the solution, add the CRISPR-Cas12a reaction system and incubate at 500 rpm for 30 min in a 37℃ constant-temperature metal shaker. Rinse three times with PBS, add 30 ng / mL HGF, and react at 37℃ for 30 min. Record the results at 0 h, 4 h, 8 h, 12 h, and 24 h. The formula for calculating the scratch migration rate (%) is:

[0081]

[0082] from Figure 11 As can be seen, compared with the HGF group, the H1 / H3 probe treated with stimulation-responsive CRISPR-Cas12a inhibited the migration ability of A549 cells at 4h, 8h, 12h, and 24h, while the H1 / H3 probe without stimulation-responsive CRISPR-Cas12a treatment had virtually no effect on cell migration. Compared with 50 nmol / L H1 / H3, the cell scratch migration rate of the 100 nmol / L H1 / H3 group treated with stimulation-responsive CRISPR-Cas12a was significantly decreased (F = 811.0, P < 0.05).

[0083] XI. Regulatory Effects on Cell Invasion Capacity

[0084] 1. Apply the matrix adhesive: Add 50 μL of diluted matrix adhesive to the upper chamber of the Transwell chamber and let it stand for 4 hours to allow it to solidify.

[0085] 2. Cell inoculation: After digestion, centrifugation and resuspending of cells, inoculate 200 μL per well and add 600 μL of culture medium to the lower chamber.

[0086] 3. Cell Intervention: Add 200 μL of serum-free culture medium to the upper chamber and 600 μL to the lower chamber, and continue culturing for 24 h. Then wash three times with PBS, incubate the prepared Td-apt with the cells for 15 min, and then add VEGF for 30 min. Taking H1 / H3 as an example, add H1 / H3 and incubate on ice for 15 min, discard the solution, and add the CRISPR-Cas12a reaction system. Incubate at 37℃ for 500 rpm for 30 min in a constant-temperature metal shaker. Wash three times with PBS, add 30 ng / mL HGF, and continue culturing at 37℃ for 24 h.

[0087] 4. Fixation and Staining: Remove the chamber, wash three times with PBS, fix with 600 μL paraformaldehyde for 30 min, then stain with 1% crystal violet solution for 20 min. Quantify cells and calculate the invasion rate using ImageJ software. The formula is:

[0088]

[0089] from Figure 11 As shown in b, compared with the HGF group, the H1 / H3 probes treated with stimulation-responsive CRISPR-Cas12a all inhibited the invasive ability of A549 cells, but the H1 / H3 probes not treated with stimulation-responsive CRISPR-Cas12a had little effect on the invasive ability of cells. Compared with 50 nmol / L H1 / H3, the cell invasion of the 100 nmol / L H1 / H3 group treated with stimulation-responsive CRISPR-Cas12a was significantly reduced (P<0.05).

[0090] Experimental Example 1

[0091] I. Optimization of Cas12a protein concentration:

[0092] 1. Cell preparation: A549 cells in good growth condition were collected after trypsin digestion, centrifuged, resuspended, and the cell density was adjusted to 1×10⁻⁶. 5 Cells / mL were seeded into confocal culture dishes and cultured in an incubator for 24 hours.

[0093] 2. Preparation of CRISPR-Cas12a reaction system: While keeping other components of the CRISPR-Cas12a reaction system unchanged, set different concentration gradients of Cas12a protein, namely 5 nmol / L, 15 nmol / L, 25 nmol / L, 35 nmol / L, and 45 nmol / L.

[0094] 3. Probe Incubation: After incubating Td-apt with A549 cells for 15 min, VEGF was added and the reaction proceeded for 30 min. Taking H1 / H3 as an example, 200 nmol / L of a mixed probe of H1 and H3 was added and incubated on ice for 15 min. After centrifugation at 1000 rpm for 3 min, the supernatant was discarded and mixed with the CRISPR-Cas12a reaction system by vortexing. The mixture was then incubated at 500 rpm for 30 min in a 37°C constant-temperature metal shaker.

[0095] 4. Fluorescence detection: The optimal reaction concentration of Cas12a protein is determined by detecting whether FRET occurs in H1 / H3 treated with the stimulus-responsive CRISPR-Cas12a system.

[0096] 5. Results: From Figure 12As can be seen, the FRET fluorescence intensity gradually increases with increasing Cas12a protein concentration. When the Cas12a protein concentration reaches 25 nmol / L, the Cas12a protein is almost completely activated. When the Cas12a protein concentration is further increased, only a slight change in FRET fluorescence intensity is observed. Therefore, the amount of Cas12a protein used in subsequent experiments is 25 nmol / L.

[0097] II. Optimization of crRNA / Cas12a protein ratio

[0098] 1. Cell preparation: The same method as for Cas12a protein concentration optimization.

[0099] 2. Preparation of CRISPR-Cas12a reaction system: While keeping other components of the CRISPR-Cas12a reaction system unchanged, set the molar ratio of crRNA / Cas12a protein to be 1:2, 2:2, 3:2, 4:2, 5:2, and 6:2.

[0100] 3. Probe incubation: The same probe incubation method as for Cas12a protein concentration optimization.

[0101] 4. Fluorescence detection: The optimal ratio of crRNA / Cas12a is determined by detecting whether FRET occurs in H1 / H3 treated by the stimulus-responsive CRISPR-Cas12a system.

[0102] 5. Results: From Figure 12 As can be seen from b, the FRET fluorescence intensity reaches its peak when the ratio of crRNA / Cas12a protein is 1:1, indicating that this ratio has been determined as the optimal ratio of crRNA / Cas12a protein.

[0103] III. Optimization of Buffer Solutions for the CRISPR-Cas12a Reaction System

[0104] 1. Cell preparation: The same method as for Cas12a protein concentration optimization.

[0105] 2. Preparation of CRISPR-Cas12a reaction system: While keeping other components of the CRISPR-Cas12a reaction system unchanged, set the buffer solutions of the CRISPR-Cas12a reaction system as NEBuffer 1, NEBuffer r2.1, NEBuffer r3.1, rCut Smart Buffer, and ExoⅠBuffer.

[0106] 3. Probe incubation: The same probe incubation method as for Cas12a protein concentration optimization.

[0107] 4. Fluorescence assay: The reaction buffer solution is determined by detecting whether FRET occurs in H1 / H3 treated with the stimulus-responsive CRISPR-Cas12a system.

[0108] 5. Results: From Figure 12 As can be seen from c, when optimizing the five buffer solutions, the FRET fluorescence intensity reached its peak when the buffer solution was NEBuffer2.1, indicating that this buffer solution was determined to be the optimal buffer solution.

[0109] IV. Optimization of Reaction Time in CRISPR-Cas12a Reaction System

[0110] 1. Cell preparation: The same method as for Cas12a protein concentration optimization.

[0111] 2. Preparation of CRISPR-Cas12a reaction system: Cas12a protein with a final concentration of 25 nmol / L, crRNA with a final concentration of 25 nmol / L, NEB buffer r2.1, and sterile water were vortexed and reacted at 37℃ for 15 min.

[0112] 3. Probe incubation: The same probe incubation method as for Cas12a protein concentration optimization.

[0113] 4. Fluorescence assay: The optimal reaction time of the CRISPR-Cas12a reaction system was determined by detecting whether FRET occurred in H1 / H3 treated by the stimulus-responsive CRISPR-Cas12a system. The assays were performed at 0 min, 10 min, 20 min, 30 min, 40 min, 50 min, and 60 min.

[0114] 5. Results: From Figure 12 As can be seen from d, when the reaction time of Cas12a protein is 30 min, the FRET fluorescence intensity increases rapidly, and only minor changes in FRET fluorescence intensity are observed in the subsequent time. Therefore, 30 min is the ideal reaction time.

[0115] V. Optimization of Reaction Temperature in CRISPR-Cas12a Reaction System

[0116] 1. Cell preparation: The same method as for Cas12a protein concentration optimization.

[0117] 2. Preparation of CRISPR-Cas12a reaction system: Cas12a protein with a final concentration of 25 nmol / L, crRNA with a final concentration of 25 nmol / L, NEB buffer r2.1, and sterile water were vortexed and reacted at 37℃ for 15 min.

[0118] 3. Probe incubation: After incubating Td-apt with cells for 15 min, add VEGF and react for 30 min. Taking H1 / H3 as an example, add 200 nmol / L of the H1 / H3 mixed probe and incubate on ice for 15 min. After centrifugation at 1000 r / min for 3 min, discard the supernatant and vortex it with the CRISPR-Cas12a reaction system. Incubate at 4℃, 25℃, 37℃, and 45℃ in a constant temperature metal shaker at 500 r / min for 30 min.

[0119] 4. Fluorescence assay: The reaction temperature of the CRISPR-Cas12a reaction system is determined by detecting whether FRET occurs in H1 / H3 treated by the stimulus-responsive CRISPR-Cas12a system.

[0120] 5. Results: From Figure 12 As can be seen from the results, within the range of 4-37℃, the FRET fluorescence signal increases with increasing reaction temperature. However, the FRET fluorescence intensity decreases significantly when the temperature exceeds 37℃. Therefore, 37℃ was selected as the optimal reaction temperature for the CRISPR-Cas12a reaction.

Claims

1. A stimulus-responsive CRISPR-Cas12a system, characterized in that, The invention comprises a DNA tetrahedron-stimulus-response factor aptamer complex and a CRISPR-Cas12a reaction system. The DNA tetrahedron-stimulus-response factor aptamer complex is formed by hybridization of a DNA tetrahedron and a stimulus-response factor aptamer. The DNA tetrahedron is formed by the self-assembly of TH1, TH2, TH3 and the target strand TS of the CRISPR-Cas12a reaction system. The stimulus-response factor aptamer hybridizes with the TS so that, under the action of a stimulus-response factor, the stimulus-response factor binds to the stimulus-response factor aptamer to expose the TS, thereby activating the CRISPR-Cas12a reaction system.

2. The stimulus-responsive CRISPR-Cas12a system according to claim 1, characterized in that, The nucleotide sequence of TH1 is shown in SEQ ID NO.1; the nucleotide sequence of TH2 is shown in SEQ ID NO.2; the nucleotide sequence of TH3 is shown in SEQ ID NO.3; the nucleotide sequence of TS is shown in SEQ ID NO.4; and the nucleotide sequence of the stimulus-response factor aptamer is shown in SEQ ID NO.

9.

3. The stimulus-responsive CRISPR-Cas12a system according to claim 1, characterized in that, The stimulus response factor is VEGF.

4. The stimulus-responsive CRISPR-Cas12a system according to claim 1, characterized in that, The Tm of the TS is greater than 40°C, and the Tm of the stimulus response factor aptamer is greater than 40°C and less than 50°C after the TS and the stimulus response factor aptamer are hybridized.

5. The stimulus-responsive CRISPR-Cas12a system according to claim 1, characterized in that, The CRISPR-Cas12a reaction system includes Cas12a protein, crRNA, and NEBuffer r2.1 buffer. The final concentration of Cas12a protein is 25 nmol / L, the final concentration of crRNA is 25 nmol / L, and the molar ratio of crRNA to Cas12a protein is 1:

1.

6. The stimulus-responsive CRISPR-Cas12a system according to claim 1, characterized in that, The molar ratio of TH1, TH2, TH3, and TS is 1:1:1:1; the molar ratio of the DNA tetrahedron to the stimulus-response aptamer is 1:

1.

7. The application of the stimulus-responsive CRISPR-Cas12a system as described in claim 1 in regulating cell membrane receptor dimerization.

8. The application of the stimulus-responsive CRISPR-Cas12a system according to claim 7, characterized in that, The regulation of cell membrane receptor dimerization refers to the regulation of c-Met receptor dimerization or c-Met receptor dimerization with TfR receptor.

9. The application of the stimulus-responsive CRISPR-Cas12a system according to claim 8, characterized in that, The steps for regulating the dimerization of c-Met receptor and TfR receptor are as follows: A549 cells are incubated with a DNA tetrahedron-stimulation response factor aptamer complex, followed by the addition of a stimulation response factor, then a mixed probe of H1 and H3 is added and incubated on ice. After centrifugation, the supernatant is removed to obtain a cell pellet. The cell pellet is then mixed with the CRISPR-Cas12a reaction system and incubated at 37°C for 30 min. The steps for regulating the dimerization of c-Met receptor and TfR receptor are as follows: A549 cells are incubated with a DNA tetrahedron-stimulation response factor aptamer complex, followed by the addition of a stimulation response factor, then a mixed probe of H1 and H2 is added and incubated on ice. After centrifugation, the supernatant is removed to obtain a cell pellet. The cell pellet is then mixed with the CRISPR-Cas12a reaction system and incubated at 37°C for 30 min. H1 and H2 are nucleic acid aptamer sequences targeting the c-Met receptor, and H3 is a nucleic acid aptamer sequence targeting the TfR receptor. The nucleotide sequence of H1 is as shown in SEQ ID. As described in NO.5, the nucleotide sequence of H2 is as described in SEQ ID NO.6, and the nucleotide sequence of H3 is as described in SEQ ID NO.7.

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