CrRNA-spacer complex, CRISPR-Cas13 system, CANTX sensor and application of crRNA-spacer complex and CRISPR-Cas13 system

Abstract

The invention discloses a crRNA-spacer complex, a CRISPR-Cas13 (Clustered Regularly Interspaced Short Palindromic Repeats-Cas13) system, a CANTX sensor and application of the CANTX sensor, and relates to the technical field of miRNA space imaging detection. A nucleotide sequence fragment of the crRNA-spacer complex provided by the invention sequentially comprises a basic fragment, a crRNA-spacer fragment and a hairpin fragment connected to the 3'end of the crRNA-spacer from the 5 'end to the 3' end; the crRNA-spacer is used for guiding a Cas13 protein in a CRISPR-Cas13 (clustered regularly interspaced short palindromic repeats-associated protein 13) system to recognize a target sequence of target miRNA (micro Ribonucleic Acid); the basic fragment is used for being combined with Cas13 protein and then loading the Cas13 protein on the surface of a delivery carrier; the hairpin fragments form a hairpin structure through complementary pairing of bases of the hairpin fragments. A CRISPR-Cas13 (clustered regularly interspaced short palindromic repeats-associated 13) system and a CANTX (Controller Area Network Temporary X) sensor, which are prepared by utilizing the crRNA-spacer complex, can be used for realizing the visual tracking of target miRNA in a cell to be detected aiming at various different miRNA.

Description

Technical Field

[0001] The present invention relates to the technical field of miRNA spatial imaging detection, and in particular to a crRNA-spacer complex, a CRISPR-Cas13 system, a CANTX sensor and applications thereof. Background Art

[0002] Myocardial infarction (MI) is a leading cause of morbidity and mortality worldwide. Cardiac fibrosis is a post-MI cardiac repair process. However, excessive cardiac fibrosis may induce adverse cardiac remodeling and eventually lead to heart failure. MiR-208 is a widely present miRNA in the heart located in the intronic region of the heavy chain myosin gene. Its significant increase is used as a supplementary indicator for the clinical diagnosis of cardiac fibrosis. MiR-208 may also directly participate in the transformation of cardiac fibroblasts induced by transforming growth factor (TGF-β), and is expected to become a new target for the preventive treatment of cardiac fibrosis. Therefore, intuitive characterization of the spatial distribution and expression level of miRNA-208 in cardiac fibroblasts will help to understand the key role of miR-208 in the process of cardiac fibrosis and regulate it, promote the benign MI cardiac repair process, and reduce the risk of heart failure in patients with myocardial infarction.

[0003] The clustered regularly interspersed short palindromic repeats (CRISPR)-Cas system is popular for its specific recognition, fast cleavage, easy operation and easy application. Its development in intracellular cardiac fibrosis-related miRNA imaging research is also very promising. At present, the CRISPR-Cas system has been successfully used for editing of multiple disease-related genes in mammals, high-throughput screening of long non-coding RNAs, live cell imaging of genomic loci and fluorescent labeling of single mRNAs. Among them, the CRISPR-Cas13 system with signal amplification characteristics has shown great value in RNA editing, in vitro analysis and disease molecular mechanism research due to its single-stranded RNA targeting ability and unique trans-enzyme cleavage activity.

[0004] However, so far, there are still limited reports on using the CRISPR-Cas13 system to achieve visual tracking of miRNA in cells. This may be related to the inhibition of Cas13 protein activity during the visual tracking of miRNA in cells by the CRISPR-Cas13 system and the poor signal leakage of RNA fluorescent reporters as trans-enzyme cleavage substrates in the complex intracellular environment. Summary of the invention

[0005] The present invention provides a crRNA-spacer complex, a CRISPR-Cas13 system, a CANTX sensor and its application. For different miRNA target sequences, the present invention optimizes the existing CRISPR-Cas13 system, extends the crRNA-spacer structure, and provides a crRNA-spacer complex to improve the system recognition efficiency. Based on this crRNA-spacer complex, the present invention also provides a CRISPR-Cas13 system, and a CANTX sensor containing a CRISPR-Cas13 system and a fluorescent reporter complex. By optimizing the continuation of the existing fluorescent reporter and increasing the Rep sequence, the optimized CANTX sensor significantly reduces the signal leakage in the physiological environment, significantly suppresses the background signal while retaining good analytical performance, and shows high sensitivity and good signal-to-noise ratio in miRNA visual tracking. The technical solution of the present invention is specifically implemented by the following technologies.

[0006] In a first aspect of the present invention, a crRNA-spacer complex is provided, wherein the nucleotide sequence fragments of the crRNA-spacer complex include, from the 5' end to the 3' end, a basic fragment, a crRNA-spacer fragment, and a hairpin fragment connected to the 3' end of the crRNA-spacer; the crRNA-spacer is used to guide the Cas13 protein in the CRISPR-Cas13 system to recognize the target sequence of the target miRNA; the basic fragment is used to bind to the Cas13 protein and then load it on the surface of the delivery vector; the hairpin fragment forms a hairpin structure through its own base complementary pairing.

[0007] Furthermore, the nucleotide sequence of the basic fragment is shown as SEQ ID NO.1, and the nucleotide sequence of the hairpin structure is shown as SEQ ID NO.2.

[0008] In the traditional CRISPR-Cas13 system used for the analysis of a selected miRNA, the length of the specially designed crRNA-spacer is the fully complementary fragment of the target sequence of the miRNA. Taking the 22nt-long miR-208 as an example, the traditional crRNA-spacer length is also 22nt.

[0009] The present invention extends the length of the 3' end of crRNA-spacer and introduces a hairpin structure, extending the original crRNA-spacer length (22nt) to 35bp. Connecting the hairpin structure at the 3' end of crRNA-spacer can effectively enhance the RNase (ribonuclease) activity of Cas13, and its catalytic constant has been tested to be as high as 107.8 / min.

[0010] The crRNA-spacer complex provided by the present invention can be used for all situations where visual tracking of target miRNA in target cells is required. For the target sequences of different target miRNAs, it is only necessary to specifically design and adjust the sequence of the crRNA-spacer fragment, and connect a hairpin structure at the 3' end of the crRNA-spacer fragment, which can effectively enhance the RNase activity of Cas13.

[0011] In the present invention, the role of the basic fragment is to bind to the Cas13 protein and load it on the carrier surface. Therefore, for different target cells and different miRNAs, the basic fragment can select a sequence that has been disclosed or undisclosed in the art and can meet the above requirements.

[0012] A second aspect of the present invention provides a product, comprising any one of the following:

[0013] (1) A CRISPR-Cas13 system comprising a Cas13 protein and any one of the above crRNA-spacer complexes.

[0014] (2) A biological material comprising any one of the above-mentioned crRNA-spacer complexes, or comprising the CRISPR-Cas13 system.

[0015] When the above-mentioned product provided by the present invention plays the same or similar functions and uses as the traditional CRISPR-Cas13 system, the RNase activity of the Cas13 protein is significantly improved compared with the existing traditional CRISPR-Cas13 system.

[0016] The third aspect of the present invention provides a CANTX (CRISPR-Cas13 based nano-toolbox) sensor, comprising the above-mentioned product, and further comprising a DNA nanoflower carrier and a fluorescent reporter complex; the nucleotide sequence fragment of the fluorescent reporter complex comprises a Rep fragment and a motif fragment in sequence from the 5' end to the 3' end;

[0017] The CRISPR-Cas13 system and the fluorescent reporter complex are loaded on the surface of the DNA nanoflower carrier, and then a layer of polymer shell is polymerized in situ to prepare the nanoflower; the polymer shell degrades in an acidic environment.

[0018] The CANTX sensor with an internal and external multi-layer structure provided by the present invention is composed of a DNA nanoflower carrier, a fluorescent reporter complex, and an optimized CRISPR-Cas13 system.

[0019] The fluorescent reporter complex can be a fluorescent reporter with a UUU motif commonly used in the art, or other fluorescent reporters with optimized motifs. Among them, DNA nanoflower vector (DNF) is used as a delivery carrier for Cas13 protein nucleic acid complexes (RNPs), and the polymer protective shell can promote cell uptake and lysosomal escape.

[0020] When the CANTX sensor provided by the present invention is used for in situ visual tracking of target miRNA in target cells, the specific action process and mechanism are as follows: the CANTX sensor enters the interior of the cell through endocytosis via the target cell; after reaching the acidic environment of the lysosome, the outermost polymer shell of the CANTX sensor is degraded and disintegrated in the acidic environment; then the CRISPR-Cas13 system (mainly the crRNA-spacer complex) is exposed to the target cell environment and released, and promotes their escape from the lysosome through the "proton sponge" effect; then, when the target miRNA exists in the target cell, the crRNA-spacer complex will continuously bind to the target miRNA target sequence in the target cell, thereby activating the Cas13 protein; the activated Cas13 protein continuously generates and amplifies the fluorescence signal intensity of the target miRNA by continuously trans-cleaving the fluorescent reporter; these fluorescent signals have a quantitative relationship with the target miRNA and can be captured by a fluorescence microscope or a fluorescence instrument, thereby reflecting the spatial position and content of the target miRNA in the target cell.

[0021] Furthermore, the nucleotide sequence of the crRNA-spacer complex and the nucleotide sequence of the fluorescent reporter complex are separated by at least 5 bases on the DNA nanoflower vector.

[0022] In order to reduce the signal leakage of CANTX sensors in physiological environments, suppress background signals, and improve the sensitivity and signal-to-noise ratio in the visual tracking of target miRNA in target cells. Furthermore, the present invention optimizes the fluorescent reporter complex. The nucleotide sequence of the motif fragment is replaced from the widely used UUU to AAU; the fluorescent reporter is modified at the 3' end of the motif fragment, and the quenching group is modified at the 5' end of the motif fragment; the sequence of the Rep fragment is complementary to the sequence of the DNA nanoflower vector.

[0023] Optionally, the sequence length of the Rep fragment is a DNA fragment of 10-15 bases.

[0024] The fluorescent reporter used in the present invention can be any one selected from fluorescent reporters commonly used in the art.

[0025] Furthermore, the fluorescent reporter is one of Cy5, Cy5.5, Cy3, Alexa Fluor 532, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 647, and FAM.

[0026] The ultimate purpose of the polymer protective shell material selected in the preparation of the CANTX sensor of the present invention is to allow the polymer protective shell to be normally degraded and disintegrated in the acidic environment of the intracellular lysosome. Therefore, as long as the polymer protective shell material can meet the above ultimate purpose and meet the safety, toxicity and other requirements in the biomedical field, it can be arbitrarily selected and used in the preparation of the CANTX sensor of the present invention.

[0027] Furthermore, the polymer protective shell is a polymer material formed by polymerizing a first monomer and a second monomer using a pH-responsive cross-linking agent; the first monomer is an acrylamide monomer; and the second monomer is an N-(3-aminopropyl)-methacrylamide monomer or an N,N-dimethylacrylamide monomer or an N,N-diethylacrylamide monomer or an N-isopropylacrylamide or an N,N-dibutylacrylamide monomer material.

[0028] DNA nanoflower vectors (DNFs) are topological flower-like nanostructures based on ultralong DNA chains and inorganic metal ion backbones. Due to their programmability, biocompatibility, and controllable assembly size for specific responses to molecular recognition stimuli, DNFs have become powerful biosensing tools for biomolecular detection.

[0029] Therefore, it can be known that the CANTX sensor provided by the present invention can use the DNA nanoflower vector commonly used in the art, for example, the DNA nanoflower vector can be prepared by the common preparation method in the art, or the related products can be directly purchased and used. For the target sequence of a specific target miRNA of a specific target cell, the corresponding crRNA-spacer is designed, so the corresponding DNA nanoflower vector can be designed in a targeted manner by using a method known and appropriate in the art.

[0030] The CANTX sensor provided by the present invention realizes the effective intracellular delivery of the CRISPR-Cas13 system (especially the crRNA-spacer complex), overcomes the problem of reduced stability of the CRISPR-Cas13 system in the complex intracellular environment and signal leakage during visual tracking, and realizes high-sensitivity visual tracking of the target miRNA. The CANTX sensor used in the present invention is a programmable miRNA visualization sensor, which further expands the intracellular application direction of the CRISPR / Cas system and provides a promising and feasible solution for studying the pathogenesis of diseases and the physiological functions of intracellular miRNA.

[0031] A fourth aspect of the present invention provides a method for preparing a CANTX sensor as described in any one of the above, comprising the following steps:

[0032] Using any DNA sequence including the basic fragment and the Rep fragment of the crRNA-spacer complex as a single-stranded DNA template to prepare a DNA nanoflower vector; mixing the DNA nanoflower vector with the fluorescent reporter complex in a HEPES buffer, removing unbound fluorescent reporter complexes, and preparing DNF / Rep nanoparticles;

[0033] The crRNA-spacer complex and Cas13 protein in the CRISPR-Cas13 system are added to a reaction buffer and mixed to prepare a Cas13 protein-nucleic acid complex;

[0034] The Cas13 protein-nucleic acid complex is mixed with the DNF / Rep nanoparticles, and reacted to prepare DNF / Rep / RNPs nanoparticles;

[0035] The DNF / Rep / RNPs nanoparticles and the polymer monomer for preparing the polymer shell are mixed and stirred evenly; a pH-responsive cross-linking agent (e.g., glycol dimethacrylate, EGDMA) is added and stirred evenly;

[0036] An initiator and an accelerator are added for reaction, and unreacted polymer monomers, pH-responsive cross-linking agent, initiator and accelerator are purified and removed to obtain the CANTX sensor.

[0037] Optionally, in the above preparation method, the molar ratio of the DNA nanoflower vector to the fluorescent reporter complex is 1:(3-5).

[0038] Optionally, in the above preparation method, the molar ratio of the DNA nanoflower vector to the fluorescent reporter complex is 1:4.

[0039] Optionally, in the above preparation method, the molar ratio of acrylamide, N-(3-aminopropyl)-methacrylamide monomer and pH-responsive cross-linking agent is (8-10):1:(1.5-3).

[0040] Optionally, in the above preparation method, the formula of HEPES buffer is 20mM HEPES, 60mM NaCl, 5mM MgCl2, pH 7.4.

[0041] Optionally, in the above preparation method, the formula of the reaction buffer is 40 mM Tris-HCl, 60 mM NaCl, 5 mM MgCl2, pH 7.4.

[0042] Optionally, in the above preparation method, the method for removing unreacted N-(3-aminopropyl)-methacrylamide monomer, initiator and promoter is: transferring the reaction product to a D-Tube Dialyzer Mini (molecular weight cutoff value is 5-14kDa) and dialyzing it in 1× PBS buffer for 6 hours to remove unreacted monomer, initiator and promoter.

[0043] A fifth aspect of the present invention provides an application of the CANTX sensor described in any one of the above items for visual tracking of target miRNA in cells to be tested.

[0044] Compared with the prior art, the present invention is beneficial in that:

[0045] 1. Based on the traditional CRISPR-Cas13 system, the present invention significantly improves the working efficiency and stability of the CRISPR-Cas13 system by extending the crRNA-spacer and designing the fluorescent reporter sequence, and provides a new strategy for the application of the CRISPR-Cas13 system in complex physiological environments.

[0046] 2. The CANTX sensor constructed in the present invention uses DNF as a carrier and is coated with an acid-responsive polymer shell on the outer layer, which significantly improves the in vivo delivery efficiency and lysosomal escape ability of the CANTX sensor, providing a good foundation for ensuring the biological function of the CRISPR-Cas13 system.

[0047] 3. The CANTX sensor constructed in the present invention exhibits high specificity, high sensitivity and high anti-interference ability in the application of intracellular miRNA tracking. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Schematic diagram of the process by which the CANTX sensor binds to the target miRNA after being endocytosed by the target cell.

[0049] Figure 2 This is the optimization process of the crRNA-spacer complex and fluorescent reporter in the CRISPR-Cas13 system. Figure 2 a is a schematic diagram of crRNA-spacer complex optimization, Figure 2 b is the activity of the CRISPR-Cas13 system before and after the optimization of the crRNA-spacer complex. Figure 2 c is a schematic diagram of fluorescence reporter optimization. Figure 2 d shows the stability results of the fluorescent reporter in PBS and culture medium containing fetal bovine serum (FBS) before and after optimization.

[0050] Figure 3 Schematic diagram of the preparation process of CANTX sensor.

[0051] Figure 4 The sensitivity and specificity test results of CANTX sensor in response to miRNA in vitro. Figure 4 a is the fluorescence signal of CANTX after incubation with target miRNA under acidic and neutral conditions, Figure 4 b is the fluorescence curve of CANTX detecting target miRNA in solution, Figure 4 c is the linear response result of CANTX detecting target miRNA in solution, Figure 4 d is the specificity evaluation result of CANTX in detecting target miRNA in solution.

[0052] Figure 5 The results of the CANTX sensor tracking miR-208 in vivo. Figure 5 a is the imaging result of CANTX sensor tracking miR-208 in cells with / without drug stimulation. Figure 5 b: Flow cytometric quantification results of CANTX sensor tracking miR-208 in cells with / without drug stimulation.

[0053] Figure 6 Toxicity evaluation results of CANTX sensor. DETAILED DESCRIPTION

[0054] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0055] In some implementation cases of the present invention, the nucleotide sequence fragment of the provided crRNA-spacer complex includes a basic fragment, a crRNA-spacer fragment and a hairpin fragment connected to the 3' end of the crRNA-spacer from the 5' end to the 3' end; the crRNA-spacer is used to guide the Cas13 protein in the CRISPR-Cas13 system to recognize the target sequence of the target miRNA; the basic fragment is used to bind to the Cas13 protein and then load it on the surface of the delivery vector; the hairpin fragment forms a hairpin structure through its own base complementary pairing.

[0056] In the traditional CRISPR-Cas13 system used for the analysis of a selected miRNA, the length of the specially designed crRNA-spacer is the fully complementary fragment of the target sequence of the miRNA. Taking the 22nt-long miR-208 as an example, the traditional crRNA-spacer length is also 22nt.

[0057] The present invention extends the length of the 3' end of crRNA-spacer and introduces a hairpin structure, extending the original crRNA-spacer length (22nt) to 35bp. Connecting a hairpin structure at the 3' end of crRNA-spacer can effectively enhance the RNase (ribonuclease) activity of Cas13, and its catalytic constant has been detected to be as high as 107.8 / min. Therefore, the crRNA-spacer complex provided by the present invention can be used for all situations where visual tracking of target miRNA in target cells is required. For target sequences of different target miRNAs, it is only necessary to design and adjust the sequence of the crRNA-spacer fragment in a targeted manner, and connect a hairpin structure at the 3' end of the crRNA-spacer fragment, which can effectively enhance the RNase activity of Cas13.

[0058] Based on the special functional effects of the above-mentioned crRNA-spacer complex, other implementation cases of the present invention also provide a product, which can be a CRISPR-Cas13 system including a Cas13 protein and any of the above-mentioned crRNA-spacer complexes. It can also be a biomaterial, including any of the above-mentioned crRNA-spacer complexes, or including the CRISPR-Cas13 system. This product can be used as a substitute for the traditional CRISPR-Cas13 system for practical application.

[0059] Other embodiments of the present invention provide a CANTX sensor, including the above product, and also including a DNA nanoflower carrier and a fluorescent reporter complex;

[0060] The specific structure of the CANTX sensor provided by the present invention is prepared by loading the CRISPR-Cas13 system and the fluorescent reporter complex on the surface of the DNA nanoflower carrier and then in situ polymerizing a layer of polymer shell; the polymer shell degrades in an acidic environment.

[0061] The CANTX sensor with an internal and external multi-layer structure provided by the present invention is composed of a DNA nanoflower carrier, a fluorescent reporter complex, and an optimized CRISPR-Cas13 system.

[0062] The fluorescent reporter complex can be a fluorescent reporter with a UUU motif commonly used in the art, or other fluorescent reporters with optimized motifs. Among them, DNA nanoflower vector (DNF) is used as a delivery carrier for Cas13 protein nucleic acid complexes (RNPs), and the polymer protective shell can promote cell uptake and lysosomal escape.

[0063] like Figure 1 As shown, the mechanism of action of the CANTX sensor provided by the present invention for achieving in situ visual tracking of the target miRNA in the target cell is as follows: (1) The CANTX sensor enters the interior of the target cell through endocytosis; (2) After reaching the acidic environment of the lysosome, the outermost polymer shell of the CANTX sensor is degraded and disintegrated in the acidic environment; (3) Then the CRISPR-Cas13 system (mainly the crRNA-spacer complex) is exposed to the target cell environment and released, and promotes their escape from the lysosome through the "proton sponge" effect; (4) Subsequently, when the target miRNA exists in the target cell, the crRNA-spacer complex will continuously bind to the target miRNA target sequence in the target cell, thereby activating the Cas13 protein; (5) The activated Cas13 protein continuously generates and amplifies the fluorescence signal intensity of the target miRNA by continuously trans-cleaving the fluorescent reporter; these fluorescent signals have a quantitative relationship with the target miRNA and can be captured by a fluorescence microscope or a fluorescence meter, thereby reflecting the spatial position and content of the target miRNA in the target cell.

[0064] Optionally, the nucleotide sequence of the crRNA-spacer complex and the nucleotide sequence of the fluorescent reporter complex are separated by at least 5 bases on the DNA nanoflower vector.

[0065] Furthermore, the present invention optimizes the fluorescent reporter complex: the nucleotide sequence fragment of the fluorescent reporter complex is designed to include a Rep fragment and a motif fragment from the 5' end to the 3' end; the nucleotide sequence of the motif fragment is replaced from the widely used UUU to AAU; the fluorescent reporter is modified at the 3' end of the motif fragment, and the quenching group is modified at the 5' end of the motif fragment; the sequence of the Rep fragment is complementary to the sequence of the DNA nanoflower vector.

[0066] After the above optimization, the signal leakage of the CANTX sensor in the physiological environment is reduced, and the background signal is suppressed, thereby showing a higher sensitivity and a better signal-to-noise ratio in the visual tracking of the target miRNA in the target cell. Optionally, the sequence length of the Rep fragment is a DNA fragment of 10 bases.

[0067] The fluorescent reporter used in the present invention can be any one selected from fluorescent reporters commonly used in the art.

[0068] Optionally, the fluorescent reporter is Cy5, Cy5.5, Cy3, Alexa Fluor 532, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 647 or FAM.

[0069] The ultimate purpose of the polymer protective shell material selected in the preparation of the CANTX sensor of the present invention is to allow the polymer protective shell to be normally degraded and disintegrated in the acidic environment of the intracellular lysosome. Therefore, as long as the polymer protective shell material can meet the above ultimate purpose and meet the safety, toxicity and other requirements in the biomedical field, it can be arbitrarily selected and used in the preparation of the CANTX sensor of the present invention.

[0070] Furthermore, the polymer protective shell is a polymer material formed by polymerizing a first monomer and a second monomer using a pH-responsive cross-linking agent; the first monomer is an acrylamide monomer; and the second monomer is an N-(3-aminopropyl)-methacrylamide monomer or an N,N-dimethylacrylamide monomer or an N,N-diethylacrylamide monomer or an N-isopropylacrylamide or an N,N-dibutylacrylamide monomer material.

[0071] DNA nanoflower vectors (DNFs) are topological flower-like nanostructures based on ultralong DNA chains and inorganic metal ion backbones. Due to their programmability, biocompatibility, and controllable assembly size for specific responses to molecular recognition stimuli, DNFs have become powerful biosensing tools for biomolecular detection.

[0072] Therefore, it can be known that the CANTX sensor provided by the present invention can use the DNA nanoflower vector commonly used in the art, for example, the DNA nanoflower vector can be prepared by the common preparation method in the art, or the related products can be directly purchased and used. For the target sequence of a specific target miRNA of a specific target cell, the corresponding crRNA-spacer is designed, so the corresponding DNA nanoflower vector can be designed in a targeted manner by using a method known and appropriate in the art.

[0073] The CANTX sensor provided by the present invention realizes the effective intracellular delivery of the CRISPR-Cas13 system (especially the crRNA-spacer complex), overcomes the reduced stability of the CRISPR-Cas13 system in the complex intracellular environment and the signal leakage problem during the visual tracking process, and realizes high-sensitivity visual tracking of the target miRNA.

[0074] Some other implementation cases of the present invention provide a method for preparing a CANTX sensor, comprising the following steps:

[0075] Using any DNA sequence including the basic fragment and the Rep fragment of the crRNA-spacer complex as a single-stranded DNA template to prepare a DNA nanoflower vector; mixing the DNA nanoflower vector with the fluorescent reporter complex in a HEPES buffer, removing unbound fluorescent reporter complexes, and preparing DNF / Rep nanoparticles;

[0076] The crRNA-spacer complex and Cas13 protein (purchased from Nanjing GenScript Co., Ltd.) in the CRISPR-Cas13 system were added to the reaction buffer and mixed to prepare a Cas13 protein-nucleic acid complex;

[0077] The Cas13 protein-nucleic acid complex is mixed with the DNF / Rep nanoparticles, and reacted to prepare DNF / Rep / RNPs nanoparticles;

[0078] The DNF / Rep / RNPs nanoparticles and acrylamide (first monomer, AAm) are mixed and stirred evenly; positively charged N-(3-aminopropyl)-methacrylamide monomer (second monomer, APMAAm) and pH-responsive crosslinking agent (e.g., glycol dimethacrylate, EGDMA) are added in sequence and stirred evenly;

[0079] An initiator and an accelerator are added for reaction, and unreacted N-(3-aminopropyl)-methacrylamide monomer, initiator and accelerator are purified and removed to obtain the CANTX sensor.

[0080] Optionally, in the above preparation method, the molar ratio of the DNA nanoflower vector to the fluorescent reporter complex is 1:(3-5).

[0081] Further optionally, the molar ratio is 1:4.

[0082] Optionally, in the above preparation method, the molar ratio of acrylamide, N-(3-aminopropyl)-methacrylamide monomer and pH-responsive cross-linking agent is (8-10):1:(1.5-3).

[0083] Further optionally, the molar ratio is 9:1:2.

[0084] Optionally, in the above preparation method, the formula of HEPES buffer is 20mM HEPES, 60mM NaCl, 5mM MgCl2, pH 7.4.

[0085] Optionally, in the above preparation method, the formula of the reaction buffer is 40 mM Tris-HCl, 60 mM NaCl, 5 mM MgCl2, pH 7.4.

[0086] Optionally, in the above preparation method, the method for removing unreacted N-(3-aminopropyl)-methacrylamide monomer, initiator and promoter is: transferring the reaction product to a D-Tube Dialyzer Mini (molecular weight cutoff value is 5-14kDa), and dialyzing in 1× PBS buffer for 6 hours to remove unreacted monomer, initiator and promoter.

[0087] The embodiment of the present invention also provides an application of the CANTX sensor described in any one of the above items, which is specifically used for visual tracking of target miRNA in cells to be tested.

[0088] Based on the optimization effect of the present invention on the crRNA-spacer complex, DNA nanoflower vector and fluorescent reporter complex, the CANTX sensor provided by the present invention can be used for all situations where visual tracking of target miRNA in target cells is required. Specifically, for the target sequence of different target miRNAs, it is only necessary to design and adjust the sequence of the crRNA-spacer fragment in a targeted manner, and connect the hairpin structure at the 3' end of the crRNA-spacer fragment, which can effectively enhance the RNase activity of Cas13.

[0089] In the following examples, miR-208 in cardiac fibroblasts was used as the target miRNA to design the crRNA-spacer complex and the corresponding CANTX sensor.

[0090] Example 1: Optimization of the CRISPR-Cas13a system in the CANTX sensor

[0091] In this example, miR-208 in cardiac fibroblasts was used as the target miRNA to design and optimize the CRISPR-Cas13a system and fluorescent reporter complex, and to form a CANTX sensor.

[0092] The optimized crRNA-spacer complex introduced a hairpin structure at the 3' end, extending the length of the crRNA-spacer, thereby increasing the crRNA-spacer to 35 bp, i.e. ACAAGCUUUUUGCUCGUCUUAUGCUAUCAGCUCUA. The crRNA-spacer complex includes a basic fragment, a crRNA-spacer fragment, and a hairpin fragment connected to the 3' end of the crRNA-spacer in sequence from the 5' end to the 3' end. The nucleotide sequence of the basic fragment is shown in SEQ ID NO.1, and the nucleotide sequence of the hairpin structure is shown in SEQ ID NO.2.

[0093] The nucleotide sequences of miR-208, traditional crRNA-spacercr, optimized crRNA-spacer complex, traditional fluorescent reporter, and optimized fluorescent reporter complex are all shown in Table 1.

[0094] The optimized fluorescent reporter complex replaces the UUU motif widely used in traditional fluorescent reporters with an AAU motif, and adds a DNA fragment complementary to DNF for 10 base pairs at its 5' end.

[0095] like Figure 2 b, After extending the length of crRNA-spacer, the trans-cleavage activity of CRISPR-Cas13a system was significant, and its catalytic constant was as high as 107.8 / min. Figure 2 As shown in d, Rep-1 is more stable under physiological conditions compared with Rep-2 composed of the UUU motif.

[0096] Table 1

[0097]

[0098] Example 2: Preparation of CANTX sensor

[0099] This example is based on the CRISPR-Cas13a system optimized in Example 1, and further uses DNF as a carrier and in situ polymerizes acid-responsive polymer nanoshells to prepare a CANTX sensor. The specific steps are as follows.

[0100] 1. Preparation of DNF

[0101] (1) Mix 1 μM single-stranded DNA template and 2 μM primer in 95 μL of 1× T4 DNA ligase buffer (40 mM Tris-HCl, 10 mM MgCl2, 10 mM dithiothreitol (DTT), 1 mM adenosine triphosphate (ATP)).

[0102] (2) Heat at 95 °C for 5 min and slowly cool to room temperature (RT) within 3 h.

[0103] (3) Add 2.5 U of T4 DNA ligase and incubate at 16°C overnight for ligation reaction.

[0104] To prepare DNF of appropriate particle size, a reaction mixture consisting of 0.5 μM ligated DNA template, 2 mM deoxyribonucleoside triphosphates (dNTPs) and 0.5 U / μL phi29 DNA polymerase was prepared at 4°C in 1 × phi29 DNA polymerase reaction buffer (50 mM Tris-HCl, 10 mM MgCl2, 10 mM ammonium sulfate, 4 mM DTT), and then reacted at 30°C for 6 h.

[0105] (4) Finally, the reaction mixture was heated to 90°C for 10 min to terminate the reaction and then quickly cooled on ice to room temperature.

[0106] The product was centrifuged at 10,000 g for 20 min and washed twice with diethyl pyrocarbonate (DEPC)-treated water to remove excess proteins and DNA chains, and then re-dissolved with an appropriate amount of DEPC-treated water.

[0107] 2. Preparation of DNF / Rep / RNPs Nanoparticles

[0108] (1) DNF and Rep were mixed at a molar ratio of 1:4 in HEPES buffer (20 mM HEPES, 60 mM NaCl, 5 mM MgCl2, pH 7.4) at 37°C for 30 min.

[0109] Unbound Rep was removed by centrifugation at 12,000 rpm for 20 min.

[0110] (2) Cas13a protein and crRNA were mixed in a reaction buffer (40 mM Tris-HCl, 60 mM NaCl, 5 mM MgCl2, pH 7.4) at a molar ratio of 1:1 and reacted at 4°C for 20 min to form Cas13a RNPs.

[0111] (3) Cas13a RNPs were reacted with previously prepared DNF / Rep nanoparticles at 4°C for 30 min to form DNF / Rep / RNPs nanoparticles.

[0112] 3. Assembly of CANTX sensor

[0113] (1) 2 μM DNF / Rep / RNPs nanoparticles were mixed with 1 M acrylamide (first monomer, AAm) solution and stirred at 4 °C for 10 min.

[0114] (2) Positively charged N-(3-aminopropyl)-methacrylamide monomer (1 M, second monomer, APMAAm) was added to the mixture and stirred at 4 °C for 10 min.

[0115] (3) The pH-responsive crosslinker ethylene glycol dimethacrylate (EGDMA, 1 M) was added to the mixture and stirred at 4 °C for 10 min. The molar ratio of AAm, APMAAm, and EGDMA was 9:1:2.

[0116] (4) Initiator ammonium persulfate (APS) and accelerator tetramethylethylenediamine (TEMED) were added to the mixture to initiate free radical polymerization and stirred at 4 °C for 2 h.

[0117] (5) The product was transferred to a D-Tube Dialyzer Mini (molecular weight cutoff 5-14 kDa) and dialyzed against 1× PBS buffer for 6 h to remove unreacted monomers, initiator, and promoter.

[0118] Example 3: In vitro response assessment of CANTX

[0119] In this example, the CANTX sensor prepared in Example 2 was subjected to an in vitro sensitivity and specificity evaluation experiment. Figure 3 As shown, the specific steps are as follows.

[0120] 1. Unlocking process of CANTX acidic response in vitro

[0121] (1) Prepare reaction buffer A (40 mM Tris-HCl, 30 mM NaCl, 30 Mm CH3COONa, 5 mM MgCl2, pH 5.4) and reaction buffer B (40 mM Tris-HCl, 60 mM NaCl, 5 mM MgCl2, pH 7.4).

[0122] (2) The CANTX sensor was first pre-reacted in reaction buffer A and reaction buffer B at 37°C for 30 min, and then the same volume of reaction buffer A or reaction buffer B containing miR-208 was added to the solution containing the CANTX sensor to activate the trans-cleavage process of Cas13a RNPs.

[0123] (3) After 60 min of reaction, the fluorescence spectra of the CANTX sensor were recorded under different pH conditions.

[0124] like Figure 4As shown in a, under acidic conditions (pH 5.4), the fluorescence intensity of the CANTX sensor is significantly greater than that under neutral conditions (pH 7.4), indicating that the CANTX sensor can undergo a self-unlocking process under acidic conditions.

[0125] 2. Sensitivity evaluation of CANTX sensor response to target miRNA

[0126] The CANTX sensor was applied to the acidic reaction buffer containing a series of different concentrations (20 pM, 50 pM, 100 pM, 500 pM, 1 nM, 2 nM and 10 nM) of miR-208, and the fluorescence signals were recorded after 1 h.

[0127] like Figure 4 As shown in (b), with the increase of miR-208 content, the fluorescence signal of CANTX sensor gradually increased. According to the fluorescence response of CANTX sensor to miR-208, the detection limit (LOD) of CANTX sensor was determined to be 20 pM.

[0128] 3. Specificity evaluation of CANTX sensor in solution

[0129] The specificity of the CANTX sensor is evaluated by:

[0130] The CANTX sensor was applied to an acidic reaction buffer containing the same concentration of miR-208 and other non-target RNAs (mis-1, miR-1, miR-21, miR-133, miR-499, the specific sequences are shown in Table 1). The fluorescence signal of the CANTX sensor was recorded after 1 h of reaction. The CANTX sensor had the strongest response to the fluorescence signal of miR-208 and almost no response to other miRNAs, indicating that the CANTX sensor has good specificity.

[0131] Example 4: Application of CANTX sensor in intracellular miR-208 tracking

[0132] 1. Cell culture and construction of myocardial fibrosis model

[0133] (1) Isolation of cardiac fibroblasts from the hearts of C57BL / 6 mice: The hearts were removed from 1-2 day old mice, minced and digested with 0.1% trypsin.

[0134] (2) Filter the cells using a 40 μm cell strainer, culture the cells for 2 h, and remove the culture medium containing cardiomyocytes.

[0135] Cells were grown in DMEM containing 10% heat-inactivated FBS, 1× antibiotics (100 U / mL penicillin and 100 μg / mL streptomycin), and 10 ng / mL bFGF. The purity of cardiac fibroblasts was confirmed by microscopic assessment and vimentin immunofluorescence analysis.

[0136] (3) Use 0.05% trypsin to separate the cells for subculture, and conduct relevant cell research at the 3rd to 4th subculture stage.

[0137] To stimulate the transformation of fibroblasts into myofibroblasts, cardiac fibroblasts were cultured in low-serum (1% FBS) DMEM medium and then cultured in low-serum medium with L-ascorbic acid (10 μg / μL) and 10 ng / mL TGF-β for 48 h.

[0138] 2. Intracellular miRNA tracking

[0139] For intracellular miR-208 tracking, the CANTX sensor was incubated with cardiac fibroblasts cultured in confocal culture dishes or 6-well plates at 37 °C for 4 h. After the 4-h incubation, the cells in the confocal culture dishes and 6-well plates were collected for confocal imaging and flow cytometry (FCM) analysis, respectively. This example shows the incubation results of CANTX with normal cardiac fibroblasts and CANTX with cardiac fibroblasts treated with drugs (TGF-β). The purpose of applying drugs is to construct a myocardial fibrosis model.

[0140] like Figure 5 As shown, CANTX can respond to the presence of miR-208 in cardiac fibroblasts, showing obvious fluorescence signals in confocal images. Compared with normal cells, the fluorescence signal of CANTX in TGF-β-treated cardiac fibroblasts was significantly enhanced, indicating that TGF-β drugs induce the expression of miR-208 in cardiac fibroblasts. Pre-treatment of cardiac fibroblasts with a specific inhibitor of miR-208 (Anti-miR) alleviated the expression of miR-208 in cardiac fibroblasts and correspondingly reduced the fluorescence signal of CANTX in cells, as shown in Figure 2. Figure 5 b shows the quantitative results of flow cytometry. The above results show that CANTX can specifically track the target miRNA in cells, and the fluorescence signal it presents is correlated with the target RNA content in cells.

[0141] Example 5: Toxicity analysis of CANTX sensor

[0142] Different concentrations of DNF, DNF / Rep, DNF / Rep / RNPs, and CANTX sensors were co-cultured with cardiac fibroblasts in a 96-well plate for 4 h. After the culture, a commercial CCK-8 kit was used to test cell activity according to the kit instructions. Figure 6 As shown, the results showed that the toxicity of CANTX sensor to cells was negligible, indicating that CANTX sensor has good biocompatibility.

[0143] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A crRNA-spacer complex, characterized in that The nucleotide sequence fragment of the crRNA-spacer complex includes a basic fragment, a crRNA-spacer fragment and a hairpin fragment connected to the 3' end of the crRNA-spacer from the 5' end to the 3' end; the crRNA-spacer is used to guide the Cas13 protein in the CRISPR-Cas13 system to recognize the target sequence of the target miRNA; the basic fragment is used to bind to the Cas13 protein and then load it on the surface of the delivery vector; the hairpin fragment forms a hairpin structure through its own base complementary pairing.

2. The crRNA-spacer complex according to claim 1, characterized in that The nucleotide sequence of the basic fragment is shown in SEQ ID NO.1, and the nucleotide sequence of the hairpin structure is shown in SEQ ID NO.

2.

3. A product, characterized in that: Include any of the following: (1) A CRISPR-Cas13 system, comprising a Cas13 protein and the crRNA-spacer complex of claim 1 or 2; (2) A biological material comprising the crRNA-spacer complex described in claim 1 or 2, or comprising the CRISPR-Cas13 system.

4. A CANTX sensor, characterized in that: The product according to claim 3 further comprises a DNA nanoflower vector and a fluorescent reporter complex; the nucleotide sequence fragment of the fluorescent reporter complex comprises a Rep fragment and a motif fragment in sequence from the 5' end to the 3' end; The CRISPR-Cas13 system and the fluorescent reporter complex are loaded on the surface of the DNA nanoflower carrier, and then a layer of polymer shell is polymerized in situ to prepare the nanoflower; the polymer shell degrades in an acidic environment.

5. The CANTX sensor according to claim 4, characterized in that: The nucleotide sequence of the crRNA-spacer complex and the nucleotide sequence of the fluorescent reporter complex are separated by at least 5 bases on the DNA nanoflower vector.

6. The CANTX sensor according to claim 4, characterized in that: The nucleotide sequence of the motif fragment is AAU; the 3' end of the motif fragment is modified with a fluorescent reporter, and the 5' end of the motif fragment is modified with a quenching group; the sequence of the Rep fragment is complementary to the sequence of the DNA nanoflower vector.

7. The CANTX sensor according to claim 6, characterized in that: The fluorescent reporter is one of Cy5, Cy5.5, Cy3, Alexa Fluor 532, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 647, and FAM.

8. The CANTX sensor according to claim 4, characterized in that: The polymer protective shell is a polymer material formed by polymerizing a first monomer and a second monomer using a pH-responsive cross-linking agent; the first monomer is an acrylamide monomer; the second monomer is an N-(3-aminopropyl)-methacrylamide monomer or an N,N-dimethylacrylamide monomer or an N,N-diethylacrylamide monomer or an N-isopropylacrylamide or an N,N-dibutylacrylamide monomer material.

9. A method for preparing the CANTX sensor according to any one of claims 4 to 8, comprising the following steps: Using any DNA sequence including the basic fragment and the Rep fragment of the crRNA-spacer complex as a single-stranded DNA template to prepare a DNA nanoflower vector; mixing the DNA nanoflower vector with the fluorescent reporter complex in a HEPES buffer, removing unbound fluorescent reporter complexes, and preparing DNF / Rep nanoparticles; The crRNA-spacer complex and Cas13 protein in the CRISPR-Cas13 system are added to a reaction buffer and mixed to prepare a Cas13 protein-nucleic acid complex; The Cas13 protein-nucleic acid complex is mixed with the DNF / Rep nanoparticles, reacted to prepare DNF / Rep / RNPs nanoparticles; The DNF / Rep / RNPs nanoparticles and the polymer monomer for preparing the polymer shell are mixed and stirred evenly; a pH-responsive cross-linking agent is added and stirred evenly; Adding an initiator and an accelerator to react, purifying and removing unreacted polymer monomers, pH-responsive cross-linking agent, initiator and accelerator to obtain the CANTX sensor; Preferably, the molar ratio of the DNA nanoflower vector to the fluorescent reporter complex is 1:(3-5); Preferably, the molar ratio of the acrylamide, N-(3-aminopropyl)-methacrylamide monomer and the pH-responsive cross-linking agent is (8-10):1:(1.5-3).

10. An application of the CANTX sensor according to any one of claims 4 to 8, characterized in that: Used for visual tracking of target miRNA in cells to be tested.

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