Nucleic acid in-situ detection system based on CRISPR / dCas fusion protein and application thereof

Abstract

The invention discloses a nucleic acid in-situ detection system based on CRISPR / dCas fusion protein and TSA signal amplification and application of the nucleic acid in-situ detection system based on CRISPR / dCas fusion protein and TSA signal amplification, and the nucleic acid in-situ detection system based on CRISPR / dCas fusion protein and TSA signal amplification comprises fusion protein based on a CRISPR / dCas system and a tyramine solution modified by a fluorophore, the fusion protein based on the CRISPR / dCas system is at least one of the following components: dCas9-HRP or biotinylated dCas9-Avi Tag, dCas9-HRP, dCas9-Avi Tag, dCas9-HRP and dCas9-Avi Wherein the dCas9-HRP has a sequence as shown in SEQ ID NO: 1 or a functional variant of the sequence as shown in SEQ ID NO: 1; the biotinylated dCas9-Avi Tag has a sequence as shown in SEQ ID NO: 2 or a functional variant of the biotinylated dCas9-Avi Tag.

Description

Technical Field

[0001] This invention relates to the field of biological gene detection technology, and in particular to an in situ nucleic acid detection system based on CRISPR / dCas fusion protein and TSA signal amplification and its application. Background Technology

[0002] In situ spatial analysis of nucleic acids in tissues is of great significance for disease research, helping pathologists to make rapid and accurate diagnoses. Currently, in situ hybridization (ISH) technology can perform qualitative, localization, and quantitative analysis in gene analysis and diagnosis, and has become the most effective molecular pathology technique. This technique uses labeled nucleic acid molecules as probes to detect DNA or RNA in cells or tissues in situ. Fluorescence in situ hybridization (FISH), using fluorescent labels, is currently the "gold standard" for detecting chromosomal ploidy and structural changes, and various commercial FISH kits have been developed and are widely used clinically. However, its clinical application is limited by drawbacks such as high experimental requirements (requiring specific instruments, such as an in situ hybridization instrument), harsh experimental conditions (requiring high-temperature denaturation at 72–95°C and treatment with carcinogenic formamide), complex procedures, long detection time, inability to detect single-base mutations, and high experimental costs. Therefore, there is an urgent need for in situ detection technologies with higher sensitivity, higher specificity, higher spatial resolution, and simple and rapid operation for spatial in situ analysis of genes in next-generation pathological sections and clinical diagnosis.

[0003] With the rapid development of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology, the high specificity (single-base sensitivity) and rapid target binding ability (within minutes to tens of minutes) of Cas proteins have attracted the attention of researchers. Because of the effectiveness of the CRISPR system in locating and editing target sites, researchers have explored methods for in-situ detection based on this system.

[0004] Existing nucleic acid in situ imaging technologies based on CRIPSR-related systems include those that combine isothermal amplification techniques to amplify detection signals and improve detection sensitivity. However, these methods suffer from cumbersome processes, high nonspecificity, and low catalytic efficiency, limiting their application. Other methods use modified long-chain sgRNAs (e.g., multiple MS2 RNA aptamers tandemly on sgRNA), but the longer sgRNAs increase the cost of their commercial synthesis. Still others use covalent modification, directly modifying dCas proteins with fluorescent groups. However, the modification process can easily damage the protein and affect its activity, and improperly processed probes can bind to intracellular proteins, generating background fluorescence. Summary of the Invention

[0005] This invention provides a nucleic acid in situ detection system based on CRISPR / dCas fusion protein and TSA signal amplification and its application, as well as two CRISPR / dCas system-based fusion proteins (dCas9-HRP and biotinylated dCas9-AviTag) for in situ detection of target genes on pathological sections and accurate pathological diagnosis.

[0006] The technical solution of the present invention is as follows: A nucleic acid in situ detection system based on CRISPR / dCas fusion protein and TSA signal amplification includes a CRISPR / dCas fusion protein and a tyramine solution modified with a fluorescent group. The CRISPR / dCas fusion protein is at least one of the following: dCas9-HRP or biotinylated dCas9-Avi Tag; wherein the dCas9-HRP has the sequence shown in SEQ ID NO: 1 or a functional variant thereof; and the biotinylated dCas9-Avi Tag has the sequence shown in SEQ ID NO: 2 or a functional variant thereof.

[0007] Preferably, in the CRISPR / dCas fusion protein and TSA signal amplification-based in situ nucleic acid detection system, the dCas9-HRP or its functional variant is encoded by the nucleic acid molecular sequence shown in SEQ ID NO: 3 or by nucleotides with more than 80% homology; the biotinylated dCas9-Avi Tag or its functional variant is encoded by the nucleic acid molecular sequence shown in SEQ ID NO: 4 or by nucleotides with more than 80% homology.

[0008] Preferably, the in-situ nucleic acid detection system based on CRISPR / dCas fusion protein and TSA signal amplification is prepared as follows: First, at least one plasmid of dCas9-HRP and dCas9-Avi Tag is constructed using the sequences of dCas9 protein, HRP protein, and Avi Tag polypeptide; then, E. coli is induced to express the fusion protein, wherein the dCas9-Avi Tag fusion protein is further recognized by biotin ligase BirA in E. coli, and biotin is linked to the lysine residue of Avi Tag through an enzymatic reaction to form a biotinylated dCas9-Avi Tag fusion protein; finally, the expressed fusion protein is purified.

[0009] Preferably, the nucleic acid in situ detection system based on CRISPR / dCas fusion protein and TSA signal amplification includes, for example, the nucleic acid in situ detection system based on the fusion protein dCas9-HRP, comprising the fusion protein dCas9-HRP and a fluorescently modified tyramine (TSA) solution; and the nucleic acid in situ detection system based on the biotinylated dCas9-Avi Tag, comprising the biotinylated dCas9-Avi Tag, streptavidin (SA) modified HRP, and a fluorescently modified tyramine (TSA) solution.

[0010] The preferred method for the in-situ nucleic acid detection system based on CRISPR / dCas fusion protein and TSA signal amplification is as follows: For dCas9-HRP, signal amplification and in-situ detection of target nucleic acids are achieved by directly catalyzing fluorescently labeled TSA; For biotinylated dCas9-Avi Tag, the dCas9-Avi Tag fusion protein is first constructed, and then recognized by the biotin ligase BirA in E. coli. Biotin is linked to the lysine residue of Avi Tag through an enzymatic reaction, and further bound to SA-HRP. The bound HRP can catalyze the signal amplification and color development of fluorescently labeled TSA.

[0011] The preferred nucleic acid in situ detection system based on CRISPR / dCas fusion protein and TSA signal amplification is the dCas9-HRP fusion protein-based nucleic acid in situ detection system, which is performed as follows: the dCas9-HRP fusion protein is incubated with the target sgRNA to assemble into a dCas9-HRP / sgRNA binary complex; the sample to be tested is contacted with the dCas9-HRP / sgRNA binary complex, incubated, and then washed; tyramine (TSA) modified with a fluorescent group is further added, and the fluorescent group covalently binds to the vicinity of the target gene under HRP catalysis; The in situ nucleic acid detection system based on the biotinylated fusion protein dCas9-Avi Tag detects nucleic acids using the following method: The biotinylated fusion protein dCas9-Avi Tag is incubated with target sgRNA to assemble into a dCas9-biotin / sgRNA binary complex; the sample to be tested is contacted with the dCas9-biotin / sgRNA binary complex, incubated, and then washed; streptavidin-modified HRP is further added, and under the specific binding of SA-biotin, HRP binds to the vicinity of the fusion protein; tyramine (TSA) modified with a fluorescent group is further added, and under the catalysis of HRP, the fluorescent group covalently binds to the vicinity of the target gene.

[0012] In the preferred embodiment of the CRISPR / dCas fusion protein and TSA signal amplification-based in-situ nucleic acid detection system, dCas9 is replaced by other dCas proteins. The three fusion proteins can be constructed not only with dCas9, but also with dCas12, dCas13, etc. However, while different dCas proteins share some common functions, they also have their own unique characteristics. For example, dCas9 / sgRNA is mainly used to recognize double-stranded DNA or single-stranded RNA, while dCas13 / sgRNA is mainly used to recognize single-stranded RNA. Those skilled in the art can decide which dCas protein to use and construct the corresponding fusion protein based on their experimental needs and target sequences.

[0013] Preferably, in the in-situ nucleic acid detection system based on CRISPR / dCas fusion protein and TSA signal amplification, the sgRNA is replaced by CrRNA or tracrRNA.

[0014] Based on the same inventive concept, the present invention also provides an application of the aforementioned nucleic acid in situ detection system based on CRISPR / dCas fusion protein and TSA signal amplification in the preparation of in situ gene detection products for pathological samples.

[0015] Based on the same inventive concept, the present invention also provides a fusion protein, wherein the fusion protein is any of the aforementioned CRISPR / dCas-based fusion proteins and the nucleic acid in situ detection system based on TSA signal amplification, which is a CRISPR / dCas-based fusion protein.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: First, in the in-situ nucleic acid detection system based on CRISPR / dCas fusion protein and TSA signal amplification of the present invention, the structural domains added to the fusion protein are specially selected, so that the method of constructing the fusion protein can be successfully combined with TSA signal amplification and applied to the in-situ nucleic acid detection system; different structural domains also endow the fusion protein with different functional activities; the fusion protein in the in-situ nucleic acid detection system based on CRISPR / dCas fusion protein and TSA signal amplification of the present invention does not require covalent modification. Compared with covalent modification, the method of constructing the fusion protein of the present invention has simple purification steps, low background interference, and improved probe specificity.

[0017] Secondly, in the in situ nucleic acid detection system based on CRISPR / dCas fusion protein and TSA signal amplification of the present invention, the significant signal amplification effect of TSA bound to the fusion protein allows researchers to observe the results using a regular fluorescence microscope, without having to rely on an expensive confocal microscope. Furthermore, in the present invention, the binding of the signal in the TSA reaction relies on the covalent bond generated by tyramine under the peroxidase reaction, thus it can withstand washing with high-strength washing solutions, which effectively reduces the interference of background fluorescence.

[0018] Third, the nucleic acid in situ detection system of the present invention is preferably a pathological section spatial in situ detection technology. Based on the efficient target recognition technology of CRISPR / dCas, the in situ detection technology of tyrosine signal amplification (TSA), and the specific affinity of SA-biotin, it has the target recognition capability of single molecule level specificity. It can realize rapid, high sensitivity, and high specificity fluorescence in situ detection of target nucleic acids, which can help clinical precision medicine and the refined level of life science research.

[0019] Of course, any product implementing this invention does not necessarily need to achieve all of the advantages described above at the same time. Attached Figure Description

[0020] Figure 1 The diagram shows a method for in situ detection of nucleic acids in pathological sections based on CRISPR / dCas fusion protein and TSA signal amplification according to the present invention.

[0021] Figure 2 The purification and Western blot characterization of three fusion proteins shown in this embodiment of the invention are as follows: dCas9-HRP, biotinylated dCas9-Avi Tag [SDS-PAGE, WB].

[0022] Figure 3 Catalytic characterization of three fusion proteins from embodiments of the present invention is shown: biotinylated dCas9-Avi Tag [purified with SA-HRP and catalyzed by DAB color development]; dCas9-HRP [catalyzed by DAB color development].

[0023] Figure 4 This invention presents experimental results of in situ nucleic acid detection based on three fusion proteins, including telomere and HER2 nucleic acid detection based on CRISPR / dCas fusion protein and TSA signal amplification. Detailed Implementation

[0024] Addressing the numerous shortcomings of existing CRIPSR-based in-situ nucleic acid imaging techniques, the inventors discovered that, on the one hand, constructing fusion proteins is a common biological method. Fusion proteins do not require covalent modification, and different functional activities can be assigned to them depending on the added domains. Compared to covalent modification, the purification process for constructing fusion proteins is simpler, with lower background interference, thus improving probe specificity. On the other hand, effective signal amplification is crucial for accurate nucleic acid detection. Tyrosine signal amplification (TSA) technology, as a highly sensitive fluorescence imaging detection method for low-abundance, difficult-to-detect targets, has become an important detection method. The significant signal amplification effect of TSA allows researchers to observe results using ordinary fluorescence microscopy, eliminating the need for expensive confocal microscopes. Furthermore, the signal binding in the TSA reaction relies on the covalent bonds generated by tyrosine under peroxidase reaction, thus allowing it to withstand washing with strong cleaning solutions, effectively reducing background fluorescence interference. Effectively combining these two technologies for successful in-situ nucleic acid imaging remains a challenge.

[0025] Based on the efficient target recognition technology of CRISPR / dCas, the in situ detection technology of tyrosine signal amplification (TSA), and the specific affinity of SA-biotin, this invention develops a nucleic acid in situ detection system based on CRISPR / dCas fusion protein and TSA signal amplification. In a preferred embodiment, the system contains at least one of two fusion proteins: dCas9-HRP or biotinylated dCas9-Avi Tag. This system is used for in situ detection and accurate pathological diagnosis of target genes on pathological sections. This nucleic acid in situ detection system has single-molecule-level specific target recognition capability, enabling the detection and imaging of target genes on pathological sections. Furthermore, it allows for single-molecule spatial in situ detection and visualization of pathogenic genes in various diseases and tumors on tissue sections, achieving accurate clinical diagnosis and efficacy monitoring of diseases and tumors. By elucidating intercellular interactions through the in situ spatial distribution information of nucleic acids, it provides a deeper understanding of the mechanisms of disease and tumor development.

[0026] This invention develops a spatial in-situ detection technology for pathological sections based on CRISPR / dCas fusion proteins and TSA signal amplification technology. This technology enables rapid, highly sensitive, and highly specific fluorescent in-situ detection of target nucleic acids, contributing to precision medicine in clinical practice and refined research in life sciences. Furthermore, this invention's nucleic acid in-situ detection system can quickly and visually detect target genes in fixed cells, exhibiting high sensitivity and specificity, thus addressing the problems of existing technologies.

[0027] A preferred and specific preparation method of the nucleic acid in situ detection system of the present invention is described in detail below: First, at least one plasmid of dCas9-HRP and dCas9-AviTag was constructed using the sequences of dCas9 protein, HRP protein, and Avi Tag polypeptide. Subsequently, E. coli was induced to express the fusion protein; among them, the dCas9-Avi Tag fusion protein was further recognized by the biotin ligase BirA in E. coli, and biotin was linked to the lysine residue of Avi Tag through an enzymatic reaction to form the biotinylated dCas9-Avi Tag fusion protein. The expressed fusion protein was then purified.

[0028] Besides dCas9 protein, the pathological slide spatial in-situ detection system based on CRISPR / dCas fusion protein and TSA signal amplification technology of the present invention can also be constructed using other dCas proteins, such as dCas12 and dCas13. Different dCas proteins share some common functions but also have their own unique characteristics. Those skilled in the art can decide which dCas protein to use and construct the corresponding fusion protein based on their experimental needs and target sequences.

[0029] The fusion protein in the in-situ nucleic acid detection system based on CRISPR / dCas fusion protein and TSA signal amplification of this invention can specifically recognize and bind to the target sequence under the guidance of sgRNA designed for the target sequence. Specifically, see... Figure 1 , dCas-HRP can directly catalyze the signal amplification of fluorescently labeled TSA and achieve in-situ detection of target nucleic acids; For the biotinylated dCas9-Avi Tag, the dCas-Avi Tag fusion protein was first constructed and then recognized by the biotin ligase BirA in E. coli. Biotin was linked to the lysine residue of the Avi Tag through an enzymatic reaction, which can further bind SA-HRP. The bound HRP can catalyze the signal amplification and color development of the fluorescently labeled TSA.

[0030] Depending on the type of tyramine molecule used in the TSA reaction, researchers can use either a fluorescence microscope or a regular optical microscope to observe the experimental results.

[0031] In this document, the range expressed as "from one value to another" is a concise way of representing a range to avoid listing all the values ​​in that range in the specification. Therefore, the description of a particular range of values ​​covers any value within that range as well as the smaller range of values ​​defined by that value, just as if the arbitrary value and the smaller range of values ​​were explicitly stated in the specification.

[0032] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Improvements and adjustments made by those skilled in the art based on the present invention in practical applications still fall within the scope of protection of the present invention.

[0033] For the sake of brevity, not all possible combinations of the technical features in each embodiment or implementation are described herein. Therefore, as long as the combinations of these technical features do not contradict each other, the technical features in each embodiment or implementation can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification. Embodiments In this embodiment, two fusion proteins based on the CRISPR / dCas system are provided, namely dCas9-HRP and biotinylated dCas9-Avi Tag, whose amino acid sequences are shown in SEQ ID NO.1-2, respectively. In this embodiment, the nucleic acid molecular sequences encoding the above fusion proteins are shown in SEQ ID NO.3-4, respectively.

[0034] The amino acid sequence of dCas9-HRP (SEQ ID NO.1) is as follows:

[0035] The amino acid sequence (SEQ ID NO.2) of the biotinylated dCas9-Avi Tag is as follows:

[0036] The nucleic acid sequences encoding the above fusion proteins are as follows: The dCas9-HRP nucleic acid molecular sequence (SEQ ID NO.3) is as follows:

[0037] The biotinylated dCas9-Avi Tag nucleic acid molecule sequence (SEQ ID NO.4) is as follows:

[0038] As an alternative embodiment, functional variants of the two fusion proteins provided by the present invention are also included within the scope of protection of the present invention. These functional variants refer to proteins that have significant or marked sequence identity or similarity compared to the parental antibodies (the two fusion proteins provided by the present invention), and that retain the biological activity of the parental fusion protein. Functional variants encompass, for example, the following variants of the fusion proteins described herein, which retain the ability to recognize target sequences to a similar, equal, or greater extent than the parental fusion protein. Referring to the parental fusion protein, the functional variant may, for example, have at least about 30%, 50%, 70%, 75%, 80%, 85%, 90%, 95%, or higher homology in amino acid sequence with the parental antibody.

[0039] Furthermore, those skilled in the art can readily employ known methods, such as directed evolution and point mutation, to mutate the nucleotide sequence corresponding to the fusion protein provided by this invention. Artificially modified nucleotides that possess 80% or more homology to the nucleotide sequence corresponding to the fusion protein described in this invention are all derived from and equivalent to the nucleotide sequence of this invention, and are also included within the scope of protection of this invention.

[0040] In the nucleic acid in situ detection system based on CRISPR / dCas fusion protein and TSA signal amplification of the present invention, the nucleic acid in situ detection method based on the fusion protein dCas9-HRP is as follows: The fusion protein dCas9-HRP was incubated with target sgRNA to assemble into a dCas9-HRP / sgRNA binary complex. The test samples were pretreated according to standard immunohistochemical procedures, then contacted with the dCas9-HRP / sgRNA binary complex, incubated, and washed. Fluorescently modified tyramine (TSA) was further added, and the fluorescent group covalently bound to the vicinity of the target gene under HRP catalysis. In situ nucleic acid detection based on the dCas9-HRP fusion protein utilizes a CRISPR recognition and TSA-mediated signal amplification strategy to locate and quantify target genes in tissue and cell specimens. The detection results can be observed using a fluorescence microscope.

[0041] In the in-situ nucleic acid detection system based on CRISPR / dCas fusion protein and TSA signal amplification of the present invention, the in-situ nucleic acid detection method based on the biotinylated fusion protein dCas9-Avi Tag is as follows: The biotinylated fusion protein dCas9-Avi Tag is incubated with target sgRNA to assemble into a dCas9-biotin / sgRNA binary complex. The test samples are pretreated according to standard immunohistochemical procedures, then contacted with the dCas9-biotin / sgRNA binary complex, incubated, and washed. Streptavidin-modified HRP is then added; under the specific binding of SA-biotin, HRP binds to the vicinity of the fusion protein. Finally, fluorescently modified tyramine (TSA) is added; under the catalysis of HRP, the fluorescent group covalently binds to the vicinity of the target gene. In situ nucleic acid detection based on the biotinylated fusion protein dCas9-Avi Tag utilizes a CRISPR recognition, SA-biotin specific binding, and TSA-mediated signal amplification strategy to locate and quantify target genes in tissue and cell specimens. The detection results can be observed using a conventional optical microscope.

[0042] The following examples of the present invention provide two fusion proteins based on the CRISPR / dCas system (dCas9-HRP and biotinylated dCas9-Avi Tag) and a nucleic acid in situ detection system based on the above fusion proteins and tyrosine signal amplification (TSA) technology, for in situ detection of target genes on pathological sections and accurate pathological diagnosis.

[0043] Example 1: Preparation of fusion proteins and their application in situ detection of telomeres and HER2 nucleic acids. The paraffin section samples from HER2-positive breast cancer patients used in this example were all obtained in accordance with relevant laws and regulations.

[0044] 1.1 Preparation and purification of fusion proteins In the following preparation method, the two fusion proteins are prepared separately, each fusion protein corresponds to a plasmid, and the respective plasmids are introduced into the E. coli system to induce expression and purify each fusion protein separately.

[0045] Specifically, First, using the sequences of dCas9 protein, HRP protein, and Avi Tag peptide, corresponding plasmids for the fusion proteins dCas9-HRP and biotinylated dCas9-Avi Tag were designed and constructed. The amino acid sequences of dCas9, HRP, and Avi Tag peptides were obtained from the Uniprot database, and after prokaryotic codon optimization, the target gene nucleotides were synthesized by Nanjing GenScript Biotech Co., Ltd. Based on the designed plasmid information, the target gene nucleotides synthesized by Nanjing GenScript Biotech Co., Ltd. were cloned into the *E. coli* pET28a expression vector to obtain plasmids encoding each fusion protein.

[0046] The plasmids encoding each fusion protein were further transformed into *E. coli* strain BL21 to induce expression of each fusion protein. After culturing, the bacterial cells were collected, sonicated, and purified by affinity chromatography to obtain the following two fusion proteins: dCas9-HRP and biotinylated dCas9-Avi Tag. For the purification and Western blot characterization of each fusion protein, please refer to [link to relevant documentation]. Figure 2 WB results showed that both fusion proteins were successfully expressed at 180 kDa, indicating that the target bands were clearly visible.

[0047] Simultaneously, the biotinylated dCas9-Avi Tag fusion protein was reacted with biotin-HRP and purified. Finally, dCas9, the biotinylated dCas9-Avi Tag, dCas9-HRP, and the biotinylated dCas9-AviTag reacted with SA-HRP were added to the TMB chromogenic reagent, respectively. It was found that dCas9-HRP and the biotinylated dCas9-AviTag reacted with SA-HRP successfully catalyzed the TMB chromogenic reaction. Figure 3 This further demonstrates the successful preparation of the two fusion proteins mentioned above.

[0048] 1.2 Binding of fusion protein to target sequence The fusion protein and sgRNA were mixed at a molar ratio of 1:2 and incubated at 37°C for 15 min to form a dCas9 fusion protein / sgRNA binary complex.

[0049] The paraffin sections were placed in xylene for 20 min, followed by 95% anhydrous ethanol for 10 min, and rinsed with deionized water to remove the wax tissue.

[0050] The dCas9 fusion protein / sgRNA binary complex was added dropwise onto a dewaxed glass slide (40 μL per cm²). The slide was incubated at 37°C for 30 min, and then washed with 1×PBS.

[0051] The aforementioned dCas9 fusion protein is selected from either dCas9-HRP or biotinylated dCas9-Avi Tag.

[0052] 1.3 Imaging based on dCas9 fusion protein 1.3.1 Imaging based on dCas9-HRP For samples used for in situ nucleic acid detection with dCas9-HRP, add tyramine modified with the fluorescent group Cy3 (TSA-Cy3) and drop it onto the glass slide (1 cm²) that has been cleaned in step 1.2. 2Add 40 μL of the slide. Incubate the slide at 37°C for 30 min, then wash the slide with 1×PBS. After washing, mount the slide and observe it under a fluorescence microscope.

[0053] 1.3.2 Imaging based on biotinylated dCas9-Avi Tag For samples used for in situ nucleic acid detection with biotinylated dCas9-Avi Tag, SA-HRP can be incubated after washing in step 1.2: Add commercially available SA-HRP, drop by drop, onto the cleaned glass slide (1 cm) from step 1.2. 2 Add 40 μl of the slide. Incubate the slide at 37°C for 30 min, then wash the slide with 1×PBS.

[0054] Then, tyramine modified with the fluorescent group AF 488 (TSA-AF 488) was added and dropped onto the glass slide that had been cleaned in the previous step (1 cm). 2 Add 40 μl of the slide. Incubate the slide at 37°C for 30 min, then wash the slide with 1×PBS. After washing, mount the slide and observe it under a fluorescence microscope.

[0055] Observation results under a fluorescence microscope are as follows Figure 4 As shown.

[0056] from Figure 4 As can be seen in: (1) The in-situ detection system based on the fusion protein dCas9-Avi Tag has been successfully implemented for detection by ordinary fluorescence microscopy; (2) The in situ detection system based on the fusion protein dCas9-HRP has successfully achieved detection by ordinary fluorescence microscopy, but the expression level is relatively low. To obtain a comparable expression level, the cost of raw materials is relatively high.

[0057] Therefore, it can be seen that the in-situ detection system based on the fusion protein dCas9-Avi Tag and the in-situ detection system based on the fusion protein dCas9-HRP provided in the above embodiments of the present invention have both successfully achieved detection by ordinary fluorescence microscopy.

[0058] Existing DNA in situ imaging techniques based on CRIPSR systems have limitations, such as cumbersome processes, high nonspecificity, and low catalytic efficiency, which restrict their application. Therefore, this invention proposes a novel approach: in situ detection using fusion proteins and TSA technology. Firstly, fusion proteins do not require covalent modification. Although constructing fusion proteins is a conventional method, different functional activities can be assigned to them depending on the added structural domains. This invention specifically selects suitable structural domains and constructs corresponding fusion proteins. Compared to covalent modification, this invention employs a fusion protein construction approach, which simplifies purification steps, reduces background interference, and improves probe specificity.

[0059] On the other hand, effective signal amplification is crucial for accurate nucleic acid detection. Tyrosine signal amplification (TSA) technology, as a highly sensitive fluorescence imaging detection method for low-abundance, difficult-to-detect targets, has become an important detection method. The significant signal amplification effect of TSA allows researchers to observe results using ordinary fluorescence microscopy, without relying on expensive confocal microscopes. Furthermore, the signal binding in the TSA reaction relies on the covalent bonds generated by tyrosine under peroxidase reaction, thus allowing it to withstand washing with strong cleaning solutions, effectively reducing interference from background fluorescence. This invention successfully combines tyrosine signal amplification (TSA) technology with fusion protein technology, achieving in-situ detection of the system using ordinary fluorescence microscopy.

[0060] The in-situ detection system based on CRISPR / dCas fusion protein and TSA provided by this invention is an emerging technology that effectively combines in-situ detection based on CRISPR / dCas fusion protein and TSA, and has the ability to bind targets quickly and with high specificity.

[0061] The present invention also has other advantages. For example, the present invention can be used to detect targets of chromosomal DNA binding to histones, which is not possible with other methods. Furthermore, the probes used in the present invention are less expensive because the present invention uses fusion proteins; therefore, the probes used are not modified long sgRNAs or covalently modified probes.

[0062] This invention provides a CRISPR / dCas9-based nucleic acid mutation detection system for use on pathological slides. To ensure the specificity of the system, the designed sgRNA was pre-aligned with the NCBI nucleic acid database to confirm that it did not have a high degree of homology with genomes including humans, animals, plants, and microorganisms.

[0063] The in situ nucleic acid detection technology for pathological sections involved in this invention can be interpreted under a regular fluorescence microscope or optical microscope, without relying on a confocal microscope. This lowers the detection threshold of the system and is conducive to its promotion to regions and hospitals with underdeveloped medical care.

[0064] This invention provides a method for in situ detection of nucleic acids on pathological sections. This method can simultaneously obtain tissue morphology and nucleic acid expression information, which can improve the accuracy of pathological diagnosis.

[0065] The pathological section in situ detection method of the present invention can be regarded as a universal detection tool, which is applicable to the detection of any nucleic acid site that meets the recognition of CRISPR / dCas9 protein. When applied to different nucleic acid detection, only the sgRNA needs to be changed.

[0066] The present invention provides an efficient in-situ detection system for CRISPR / dCas fusion proteins and TSA, offering numerous advantages. The inventors believe this technology will be widely applied in the future and will provide strong support for research in related fields.

[0067] Under the guidance of the present invention and the above embodiments, those skilled in the art will readily foresee that all the raw materials or their equivalents, processing methods or their equivalents listed or exemplified in the present invention can achieve the present invention, and that the upper and lower limits and range values ​​of the parameters of each raw material and processing method can also achieve the present invention. Examples are not listed one by one here.

Claims

1. A nucleic acid in situ detection system based on CRISPR / dCas fusion protein and TSA signal amplification, characterized in that, The invention comprises a fusion protein based on a CRISPR / dCas system and a tyramine solution modified with a fluorescent group, wherein the fusion protein based on the CRISPR / dCas system is at least one of the following: dCas9-HRP or biotinylated dCas9-Avi Tag; wherein the dCas9-HRP is the sequence shown in SEQ ID NO: 1; the biotinylated dCas9-Avi Tag is the sequence shown in SEQ ID NO: 2; wherein the nucleic acid in situ detection system based on the fusion protein dCas9-HRP comprises the fusion protein dCas9-HRP and a tyramine (TSA) solution modified with a fluorescent group; and the nucleic acid in situ detection system based on the biotinylated dCas9-Avi Tag comprises the biotinylated dCas9-Avi Tag, streptavidin (SA) modified HRP, and a tyramine (TSA) solution modified with a fluorescent group; and, The nucleic acid in situ detection system based on the fusion protein dCas9-HRP detects nucleic acids using the following method: the fusion protein dCas9-HRP is incubated with the target sgRNA to assemble into a dCas9-HRP / sgRNA binary complex; the sample to be tested is contacted with the dCas9-HRP / sgRNA binary complex, incubated, and then washed; tyramine (TSA) modified with a fluorescent group is further added, and the fluorescent group covalently binds to the vicinity of the target gene under HRP catalysis. The in situ nucleic acid detection system based on the biotinylated fusion protein dCas9-Avi Tag detects nucleic acids using the following method: The biotinylated fusion protein dCas9-Avi Tag is incubated with target sgRNA to assemble into a dCas9-biotin / sgRNA binary complex; the sample to be tested is contacted with the dCas9-biotin / sgRNA binary complex, incubated, and then washed; streptavidin-modified HRP is further added, and under the specific binding of SA-biotin, HRP binds to the vicinity of the fusion protein; tyramine (TSA) modified with a fluorescent group is further added, and under the catalysis of HRP, the fluorescent group covalently binds to the vicinity of the target gene.

2. The in-situ nucleic acid detection system based on CRISPR / dCas fusion protein and TSA signal amplification as described in claim 1, characterized in that, The dCas9-HRP is encoded by the nucleic acid sequence shown in SEQ ID NO: 3; the biotinylated dCas9-Avi Tag is encoded by the nucleic acid sequence shown in SEQ ID NO:

4.

3. The in-situ nucleic acid detection system based on CRISPR / dCas fusion protein and TSA signal amplification as described in claim 1, characterized in that, The preparation of the CRISPR / dCas fusion protein is as follows: First, at least one plasmid of dCas9-HRP and dCas9-Avi Tag is constructed using the sequences of dCas9 protein, HRP protein, and Avi Tag polypeptide. Then, E. coli is induced to express the fusion protein. The dCas9-Avi Tag fusion protein is further recognized by the biotin ligase BirA in E. coli, and biotin is linked to the lysine residue of Avi Tag through an enzymatic reaction to form the biotinylated dCas9-Avi Tag fusion protein. Finally, the expressed fusion protein is purified.

4. The in-situ nucleic acid detection system based on CRISPR / dCas fusion protein and TSA signal amplification as described in claim 1, characterized in that, For dCas9-HRP, signal amplification and in-situ detection of target nucleic acids are achieved by directly catalyzing fluorescently labeled TSA. For biotinylated dCas9-Avi Tag, a dCas9-Avi Tag fusion protein is first constructed and then recognized by the biotin ligase BirA in E. coli. Biotin is linked to the lysine residue of Avi Tag through an enzymatic reaction, and then SA-HRP is further bound. The bound HRP can catalyze the signal amplification and color development of fluorescently labeled TSA.

5. The in-situ nucleic acid detection system based on CRISPR / dCas fusion protein and TSA signal amplification as described in claim 1, characterized in that, The sgRNA is replaced by CrRNA or tracrRNA.

6. The application of the nucleic acid in situ detection system based on CRISPR / dCas fusion protein and TSA signal amplification as described in any one of claims 1-5 in the preparation of in situ gene detection products for pathological samples.

7. A fusion protein, characterized in that, The fusion protein based on the CRISPR / dCas system in the nucleic acid in situ detection system based on CRISPR / dCas fusion protein and TSA signal amplification as described in any of claims 1-5.

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