In situ nucleic acid detection system based on CRISPR / dCas fusion protein and TSA signal amplification and its application

Abstract

The present invention discloses an in situ nucleic acid detection system based on CRISPR / dCas fusion protein and TSA signal amplification and an application thereof. The in situ nucleic acid detection system based on CRISPR / dCas fusion protein and TSA signal amplification comprises a fusion protein based on the CRISPR / dCas system and a fluorescent group-modified tyramide solution. The fusion protein based on the CRISPR / dCas system is at least one of the following: dCas9-HRP, dCas9-Avidin, or biotinylated dCas9-Avi Tag; wherein the dCas9-HRP has the sequence shown in SEQ ID NO: 1 or a functional variant thereof; the dCas9-Avidin has the sequence shown in SEQ ID NO: 2 or a functional variant thereof; and the biotinylated dCas9-Avi Tag has the sequence shown in SEQ ID NO: 3 or a functional variant thereof.

Description

Technical Field

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

[0002] Spatial in situ analysis of nucleic acids in tissues is of great significance for disease research, aiding pathologists in rapid and accurate diagnosis. Currently, in situ hybridization (ISH) has become the most effective molecular pathology technique, enabling qualitative, localized, and quantitative analysis in genetic analysis and diagnosis. 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 fluorophores as labels, is currently the gold standard for detecting chromosome ploidy and structural changes. Numerous commercial FISH kits have been developed and are widely used clinically. However, its clinical application is limited by its demanding experimental conditions (requiring specialized instrumentation, such as an in situ hybridization instrument), demanding experimental conditions (requiring high-temperature denaturation at 72–95°C and treatment with the carcinogen formamide), complex protocols, prolonged 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, simple and rapid operation for the spatial in situ analysis and clinical diagnosis of genes in the next generation of pathological sections.

[0003] With the rapid development of CRISPR (Clustered regularly interspaced short palindromic repeats) technology, the high specificity (single-base sensitivity) and rapid target binding (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] There are existing nucleic acid in situ imaging technologies based on CRIPSR-related systems. Some combine isothermal amplification technology to amplify detection signals and improve detection sensitivity, but this solution has problems such as cumbersome process, high non-specificity, and low catalytic efficiency, which limit its application; some use modified long-chain sgRNA (for example, multiple MS2 RNA aptamers are connected in series on the sgRNA), but the longer sgRNA increases the cost of its commercial synthesis; some use covalent modification to directly modify the fluorescent group on the dCas protein, but the modification process is prone to protein loss and affect protein activity, and uncleaned probes will bind to proteins in the cell and produce background fluorescence. Summary of the Invention

[0005] The present invention provides an in situ nucleic acid detection system based on CRISPR / dCas fusion protein and TSA signal amplification and its application, as well as three fusion proteins based on the CRISPR / dCas system (dCas9-HRP, dCas9-Avidin, and biotinylated dCas9-Avi Tag), which are used for in situ detection of target genes on pathological sections and precise pathological diagnosis.

[0006] The technical solutions of the present invention are as follows:

[0007] A nucleic acid in situ detection system based on CRISPR / dCas fusion protein and TSA signal amplification, comprising a fusion protein based on the CRISPR / dCas system and a fluorescent group-modified tyramide solution, wherein the fusion protein based on the CRISPR / dCas system is at least one of the following: dCas9-HRP, dCas9-Avidin, or biotinylated dCas9-Avi Tag; wherein the dCas9-HRP has the sequence shown in SEQ ID NO: 1 or a functional variant thereof; the dCas9-Avidin has the sequence shown in SEQ ID NO: 2 or a functional variant thereof; and the biotinylated dCas9-Avi Tag has the sequence shown in SEQ ID NO: 3 or a functional variant thereof.

[0008] The nucleic acid in situ detection system based on CRISPR / dCas fusion protein and TSA signal amplification is preferably such that the dCas9-HRP or a functional variant thereof is encoded by a nucleic acid molecule sequence shown in SEQ ID NO: 4 or a nucleotide with more than 80% homology; the dCas9-Avidin or a functional variant thereof is encoded by a nucleic acid molecule sequence shown in SEQ ID NO: 5 or a nucleotide with more than 80% homology; the biotinylated dCas9-Avi Tag or a functional variant thereof is encoded by a nucleic acid molecule sequence shown in SEQ ID NO: 6 or a nucleotide with more than 80% homology.

[0009] The nucleic acid in situ detection system based on CRISPR / dCas fusion protein and TSA signal amplification is preferred. The preparation of the fusion protein based on the CRISPR / dCas system is as follows: first, using the sequences of dCas9 protein, HRP protein, Avidin protein, and Avi Tag polypeptide, at least one plasmid of dCas9-HRP, dCas9-Avidin, and dCas9-Avi Tag is constructed; then, Escherichia coli is induced to express the fusion protein, wherein the dCas9-Avi Tag fusion protein is further recognized by the biotin ligase BirA in Escherichia 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.

[0010] The nucleic acid in situ detection system based on CRISPR / dCas fusion protein and TSA signal amplification is preferably: the nucleic acid in situ detection system based on the fusion protein dCas9-HRP includes the fusion protein dCas9-HRP and a fluorescent group-modified tyramide (TSA) solution; the nucleic acid in situ detection system based on the fusion protein dCas9-Avidin includes the fusion protein dCas9-Avidin, biotinylated HRP, and a fluorescent group-modified tyramide (TSA) solution; the nucleic acid in situ detection system based on the biotinylated dCas9-Avi Tag includes a biotinylated dCas9-Avi Tag, streptavidin (SA)-modified HRP, and a fluorescent group-modified tyramide (TSA) solution.

[0011] The nucleic acid in situ detection system based on CRISPR / dCas fusion protein and TSA signal amplification is preferably: for dCas9-HRP, signal amplification and in situ detection of target nucleic acid are achieved by directly catalyzing TSA labeled with fluorescent molecules; for dCas9-Avidin, biotin-HRP is first combined, and the bound HRP catalyzes TSA labeled with fluorescent molecules for signal amplification and color development; for biotinylated dCas9-Avi Tag, dCas9-Avi Tag fusion protein is first constructed, and further recognized by biotin ligase BirA in Escherichia coli, biotin is linked to the lysine residue of Avi Tag through an enzymatic reaction, and further combined with SA-HRP, and the bound HRP can catalyze TSA labeled with fluorescent molecules for signal amplification and color development.

[0012] The nucleic acid in situ detection system based on CRISPR / dCas fusion protein and TSA signal amplification is preferably a nucleic acid in situ detection system based on the fusion protein dCas9-HRP, which is detected by 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; and fluorescent group-modified tyramide (TSA) is further added, and the fluorescent group is covalently bound to the vicinity of the target gene under the catalysis of HRP;

[0013] The nucleic acid in situ detection system of the fusion protein dCas-Avidin is detected by the following method: the fusion protein dCas9-Avidin is incubated with the target sgRNA to assemble into a dCas9-Avidin / sgRNA binary complex; the sample to be tested is contacted with the dCas9-Avidin / sgRNA binary complex, incubated and then washed; biotinylated HRP is further added, and under the specific binding of avidin-biotin, HRP binds to the vicinity of the fusion protein; fluorophore-modified tyramide (TSA) is further added, and the fluorophore is covalently bound to the vicinity of the target gene under the catalysis of HRP;

[0014] The nucleic acid in situ detection system based on the biotinylated fusion protein dCas9-Avi Tag is detected by the following method: the biotinylated fusion protein dCas9-Avi Tag is incubated with the 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; HRP modified with streptavidin SA is further added, and under the specific binding of SA-biotin, HRP binds to the vicinity of the fusion protein; tyramide modified with a fluorescent group (TSA) is further added, and the fluorescent group is covalently bound to the vicinity of the target gene under the catalysis of HRP.

[0015] The nucleic acid in situ detection system based on CRISPR / dCas fusion protein and TSA signal amplification is preferred, and the dCas9 is replaced by other dCas proteins. The three fusion proteins can be constructed not only with dCas9, but also with dCas12, dCas13, etc., which can construct corresponding fusion proteins. However, different dCas proteins have their own characteristics in addition to some common functions. For example, dCas9 / sgRNA is mainly used to recognize double-stranded DNA or single-stranded RNA, and dCas13 / sgRNA is mainly used to recognize single-stranded RNA. Those skilled in the art can decide which dCas protein to use according to their own experimental requirements and target sequences, and thus construct the corresponding fusion protein.

[0016] The nucleic acid in situ detection system based on CRISPR / dCas fusion protein and TSA signal amplification is preferred, wherein the sgRNA is replaced by crRNA or tracrRNA.

[0017] 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.

[0018] Based on the same inventive concept, the present invention also provides a fusion protein, which is a fusion protein based on the CRISPR / dCas system in any of the aforementioned nucleic acid in situ detection systems based on CRISPR / dCas fusion protein and TSA signal amplification.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] First, in the nucleic acid in situ detection system based on CRISPR / dCas fusion protein and TSA signal amplification of the present invention, the present invention makes a special selection of the structural domain added to the fusion protein, so that the method of constructing the fusion protein is successfully combined with TSA signal amplification and applied to the nucleic acid in situ detection system; different structural domains also give the fusion protein different functional activities; the fusion protein in the nucleic acid in situ 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 scheme for constructing the fusion protein of the present invention has simple purification steps, low background interference, and improved probe specificity.

[0021] Second, in the nucleic acid in situ 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 through an ordinary fluorescence microscope without having to rely on expensive confocal microscopes; and the binding of signals in the TSA reaction of the present invention relies on the covalent bond generated by tyramide under the peroxidase reaction, so it can withstand washing with high-intensity cleaning solutions, which effectively reduces the interference of background fluorescence.

[0022] Third, the nucleic acid in situ detection system of the present invention is preferably a spatial in situ detection technology for pathological sections, based on the efficient target recognition technology of CRISPR / dCas, the in situ detection technology of tyramide signal amplification (TSA), and the specific affinity of SA-biotin. It has single-molecule-level specific target recognition capabilities and can achieve rapid, highly sensitive, and highly specific fluorescent in situ detection of target nucleic acids, thereby facilitating refined research in clinical precision medicine and life sciences.

[0023] Of course, any product implementing the present invention does not necessarily need to achieve all of the advantages described above at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Shown is a schematic diagram of a method for in situ detection of nucleic acids in pathological sections based on CRISPR / dCas fusion protein and TSA signal amplification of the present invention.

[0025] Figure 2 The purification and WB characterization of three fusion proteins of the present invention are shown: dCas9-HRP, dCas9-Avidin, and biotinylated dCas9-Avi Tag [SDS-PAGE, WB].

[0026] Figure 3 The catalytic characterization of three fusion proteins of the present invention is shown: dCas9-Avidin [purified with biotin-HRP and catalyzed by DAB color development]; biotinylated dCas9-Avi Tag [purified with SA-HRP and catalyzed by DAB color development]; dCas9-HRP [catalyzed by DAB color development].

[0027] Figure 4 The experimental results of the in situ detection of nucleic acids based on three fusion proteins in the embodiment of the present invention are shown, including in situ detection of telomeres and HER2 nucleic acids based on CRISPR / dCas fusion protein and TSA signal amplification. DETAILED DESCRIPTION

[0028] Based on the numerous shortcomings of existing in situ nucleic acid imaging technologies based on CRIPSR systems, the inventors discovered that, on the one hand, constructing fusion proteins is a commonly used biological method that does not require covalent modification. Fusion proteins can be endowed with different functional activities depending on the added domains. Compared with covalent modification, constructing fusion proteins requires simpler purification steps, reduces background interference, and improves probe specificity. On the other hand, effective signal amplification is key to achieving accurate nucleic acid detection. Tyramide signal amplification (TSA) technology has become an important method for highly sensitive fluorescence imaging of low-abundance and difficult-to-detect targets. Its significant signal amplification allows researchers to observe results using conventional fluorescence microscopy, eliminating the need for expensive confocal microscopes. Furthermore, signal binding in the TSA reaction relies on the covalent bond formed by tyramide in the peroxidase reaction, making it resistant to high-intensity washing solutions, effectively reducing background fluorescence interference. The challenge remains to effectively combine these two technologies for successful in situ nucleic acid imaging.

[0029] Based on the efficient target recognition technology of CRISPR / dCas, the in situ detection technology of tyramide signal amplification (TSA), and the specific affinity of SA-biotin, the present invention has developed a nucleic acid in situ detection system based on CRISPR / dCas fusion protein and TSA signal amplification. In a preferred exemplary embodiment, the system contains at least one of three fusion proteins: dCas9-HRP, dCas9-Avidin, or biotinylated dCas9-Avi Tag, for in situ detection and precise pathological diagnosis of target genes on pathological sections. This nucleic acid in situ detection system has single-molecule-level specific target recognition capabilities, can detect and image target genes on pathological sections, and then perform single-molecule spatial in situ detection and visualization of pathogenic genes in various diseases and tumors on tissue sections, thereby achieving precise clinical diagnosis and efficacy monitoring of diseases and tumors, and elucidating cell-to-cell interactions through the in situ distribution information of nucleic acid space, deeply revealing the mechanisms of disease and tumorigenesis.

[0030] The spatial in situ detection technology for pathological sections developed by the present invention, based on CRISPR / dCas fusion proteins and TSA signal amplification technology, can achieve rapid, highly sensitive, and highly specific fluorescence in situ detection of target nucleic acids, assisting in the refinement of clinical precision medicine and life science research. Furthermore, the nucleic acid in situ detection system of the present invention can quickly and visually detect target genes in situ in fixed cells, with high sensitivity and specificity, and can solve the problems of the existing technology.

[0031] A preferred and specific preparation method of the nucleic acid in situ detection system of the present invention is described in detail as follows:

[0032] First, at least one plasmid of dCas9-HRP, dCas9-Avidin, or dCas9-Avi Tag is constructed using the sequences of dCas9 protein, HRP protein, Avidin protein, and Avi Tag polypeptide;

[0033] Afterwards, 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 attached to the lysine residue of Avi Tag through an enzymatic reaction to form a biotinylated dCas9-Avi Tag fusion protein;

[0034] Subsequently, the expressed fusion protein was purified.

[0035] In addition to the dCas9 protein, the pathology section 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 have their own characteristics in addition to some common functions. Those skilled in the art can decide which dCas protein to use based on their experimental requirements and target sequence, and thus construct the corresponding fusion protein.

[0036] The fusion protein in the nucleic acid in situ detection system based on CRISPR / dCas fusion protein and TSA signal amplification of the present invention can specifically recognize and bind to the target sequence under the guidance of the sgRNA designed for the target sequence. Figure 1 ,

[0037] For dCas-HRP, it can directly catalyze fluorescent molecule-labeled TSA to amplify the signal and realize in situ detection of target nucleic acids;

[0038] For dCas-Avidin, it needs to be combined with biotin-HRP first. The combined HRP can catalyze the fluorescent molecule-labeled TSA to amplify the signal and develop color.

[0039] For biotinylated dCas9-Avi Tag, a dCas-Avi Tag fusion protein is first constructed and further recognized by the biotin ligase BirA in Escherichia coli. Biotin is then linked to the lysine residue of the Avi Tag through an enzymatic reaction, which can then be further bound to SA-HRP. The bound HRP can catalyze the fluorescent molecule-labeled TSA for signal amplification and color development.

[0040] Depending on the tyramide molecule used in the TSA reaction, researchers can observe the experimental results using either a fluorescence microscope or a regular light microscope.

[0041] In this document, the term "from a value to another value" is used as a summary to avoid listing all values ​​within the range. Therefore, a description of a specific numerical range encompasses any value within that range and any smaller numerical ranges defined by any value within that range, just as if the values ​​and smaller numerical ranges were explicitly stated in the specification.

[0042] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only intended to illustrate the present invention and are not intended to limit the scope of protection of the present invention. Improvements and adjustments made by those skilled in the art in accordance with the present invention in actual applications still fall within the scope of protection of the present invention.

[0043] In this document, for the sake of simplicity, not all possible combinations of the various technical features in each embodiment or example are described. Therefore, as long as there is no contradiction in the combination of these technical features, the various technical features in each embodiment or example can be combined arbitrarily, and all possible combinations should be considered to be within the scope of this specification.

[0044] In this embodiment, three fusion proteins based on the CRISPR / dCas system are first provided, namely dCas9-HRP, dCas9-Avidin, and biotinylated dCas9-Avi Tag, whose amino acid sequences are shown in SEQ ID NO.1-3, respectively; in this embodiment, the nucleic acid molecule sequences encoding the above fusion proteins are shown in SEQ ID NO.4-6, respectively.

[0045] Specifically, the dCas9-HRP amino acid sequence is as follows:

[0046]

[0047] The amino acid sequence of dCas9-Avidin is as follows:

[0048]

[0049] The amino acid sequence of dCas9-Avi is as follows:

[0050]

[0051] The nucleic acid sequences encoding the above fusion proteins are as follows:

[0052] dCas9-HRP nucleic acid molecule sequence:

[0053]

[0054] dCas9-Avidin nucleic acid molecule sequence:

[0055]

[0056] dCas9-Avi Tag nucleic acid molecule sequence:

[0057]

[0058] As an alternative embodiment, the functional variants of the above-mentioned three fusion proteins provided by the present invention are also included in the protection scope of the present invention, and the functional variants refer to proteins with obvious or significant sequence identity or similarity compared to the parent antibody (three fusion proteins provided by the present invention), and the functional variants retain the biological activity of the parent fusion protein. Functional variants encompass the following variants of fusion proteins such as those described herein, which retain the ability to identify target sequences to a similar extent, to the same extent, or to a higher degree than the parent fusion protein. With reference to the parent fusion protein, functional variants can be, for example, having at least about 30%, 50%, 70%, 75%, 80%, 85%, 90%, 95% or higher homology in amino acid sequence with the parent antibody.

[0059] Furthermore, those skilled in the art can readily mutate the nucleotide sequence corresponding to the fusion protein provided herein using known methods, such as directed evolution and point mutagenesis. Artificially modified nucleotide sequences that share 80% or greater homology with the nucleotide sequence corresponding to the fusion protein provided herein are derived from and are equivalent to the nucleotide sequence provided herein and are also encompassed within the scope of protection of the present invention.

[0060] Among them, 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:

[0061] The fusion protein dCas9-HRP is incubated with the target sgRNA to form a dCas9-HRP / sgRNA binary complex. The sample to be tested is pretreated according to standard immunohistochemistry procedures and then exposed to the dCas9-HRP / sgRNA binary complex, incubated, and washed. Fluorophore-modified tyramide (TSA) is then added, and the fluorescent group covalently binds to the target gene under HRP catalysis. In situ nucleic acid detection based on the fusion protein dCas9-HRP uses CRISPR recognition and TSA-mediated signal amplification to locate and quantify target genes in tissue and cell specimens. The results can be observed using a fluorescence microscope.

[0062] 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-Avidin is as follows:

[0063] The fusion protein dCas9-Avidin is incubated with the target sgRNA to form a dCas9-Avidin / sgRNA binary complex. The sample to be tested is pretreated according to standard immunohistochemistry procedures and then exposed to the dCas9-Avidin / sgRNA binary complex, incubated, and washed. Biotinylated HRP is then added, and the specific binding of avidin-biotin causes HRP to bind to the vicinity of the fusion protein. Fluorophore-modified tyramide (TSA) is then added, and the fluorescent group covalently binds to the target gene under HRP catalysis. In situ nucleic acid detection based on the fusion protein dCas9-Avidin utilizes CRISPR recognition, specific binding of avidin-biotin, and TSA-mediated signal amplification to localize and quantify target genes in tissue and cell specimens. The results can be observed using a standard optical microscope.

[0064] 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 biotinylated fusion protein dCas9-Avi Tag is as follows:

[0065] The biotinylated fusion protein dCas9-Avi Tag is incubated with the target sgRNA to form a dCas9-biotin / sgRNA binary complex. The sample to be tested is pretreated according to standard immunohistochemistry procedures and then exposed to the dCas9-biotin / sgRNA binary complex, incubated, and washed. Streptavidin (SA)-modified HRP is then added. Under the specific binding of SA-biotin, HRP binds to the vicinity of the fusion protein. Furthermore, fluorophore-modified tyramide (TSA) is added, and under HRP catalysis, the fluorophore covalently binds to the vicinity of the target gene. In situ nucleic acid detection based on the biotinylated fusion protein dCas9-Avi Tag utilizes CRISPR recognition, SA-biotin specific binding, and TSA-mediated signal amplification to localize and quantify target genes in tissue and cell specimens. The results can be observed using a standard optical microscope.

[0066] The following examples of the present invention provide three fusion proteins based on the CRISPR / dCas system (dCas9-HRP, dCas9-Avidin, and biotinylated dCas9-Avi Tag) and an in situ nucleic acid detection system based on the above fusion proteins and tyramide signal amplification (TSA) technology, which are used for in situ detection of target genes on pathological sections and precise pathological diagnosis.

[0067] Example 1: Preparation of fusion protein and its application in in situ detection of telomere and HER2 nucleic acid

[0068] The paraffin sections of HER2-positive breast cancer patients used in this example were obtained in accordance with relevant laws and regulations.

[0069] 1.1 Preparation and purification of fusion protein

[0070] In the following preparation method, three fusion proteins are prepared separately. Each fusion protein corresponds to a plasmid. Each plasmid is introduced into the E. coli system, and each fusion protein is induced, expressed, and purified separately.

[0071] Specifically,

[0072] First, using the sequences of dCas9 protein, HRP protein, Avidin protein, and Avi Tag polypeptide, corresponding plasmids were designed to construct the fusion proteins dCas9-HRP, dCas9-Avidin, and biotinylated dCas9-Avi Tag, respectively. The amino acid sequences of dCas9, HRP, Avidin protein, and Avi Tag polypeptide were obtained from the Uniprot database. After prokaryotic codon optimization, the nucleotides of the target genes were commissioned to Nanjing GenScript for synthesis. Based on the designed plasmid information, the nucleotides of the target genes synthesized by Nanjing GenScript were then cloned into the Escherichia coli pET28a expression vector to obtain plasmids encoding each of the fusion proteins.

[0073] The plasmids encoding each fusion protein were further transformed into E. coli BL21 strains, and the expression of each fusion protein was induced. After cultivation, the cells were collected, ultrasonically disrupted, and purified using affinity chromatography to obtain the following three fusion proteins: dCas9-HRP, dCas9-Avidin, and biotinylated dCas9-Avi Tag. For purification and western blotting characterization of the three fusion proteins, see Figure 2 , WB results showed that obvious target bands appeared at 180KDa, indicating the successful expression of the three fusion proteins.

[0074] At the same time, dCas9-Avidin and biotinylated dCas9-Avi Tag fusion proteins were reacted with biotin-HRP and SA-HRP, respectively, and purified. Finally, dCas9, dCas9-Avidin, biotinylated dCas9-Avi Tag, dCas9-HRP, dCas9-Avidin reacted with biotin-HRP, and biotinylated dCas9-Avi Tag reacted with SA-HRP were added to TMB colorimetric reagent, respectively. It was found that dCas9-HRP, dCas9-Avidin, and biotinylated dCas9-Avi Tag reacted with SA-HRP could successfully catalyze the TMB colorimetric reaction ( Figure 3 ), further indicating the successful preparation of the above three fusion proteins.

[0075] 1.2 Binding of fusion protein to target sequence

[0076] 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.

[0077] The paraffin sections were placed in xylene for 20 min, then in 95% anhydrous ethanol for 10 min, and rinsed with deionized water to remove the wax tissue.

[0078] The dCas9 fusion protein / sgRNA binary complex was added dropwise onto a dewaxed glass slide (40 μL per 1 cm2). The slides were incubated at 37°C for 30 min and then washed with 1× PBS.

[0079] The above-mentioned dCas9 fusion protein is selected from any one of dCas9-HRP, dCas9-Avidin, and biotinylated dCas9-AviTag.

[0080] 1.3 Imaging based on dCas9 fusion proteins

[0081] 1.3.1 dCas9-HRP-based imaging

[0082] For samples using dCas9-HRP for in situ nucleic acid detection, add tyramide modified with the fluorescent group Cy3 (TSA-Cy3) and drop it onto the glass slide cleaned in 1.2 (1 cm 2 Add 40 μL of the solution dropwise. Incubate the slides at 37°C for 30 minutes, then wash with 1× PBS. After washing, mount the slides and observe under a fluorescence microscope.

[0083] 1.3.2 dCas9-Avidin-based Imaging

[0084] For samples using dCas9-Avidin for in situ nucleic acid detection, biotin-HRP can be incubated after washing in step 1.2:

[0085] Add commercially purchased biotin-HRP and drop it onto the glass slide (1 cm 2 40 μl was added dropwise). The slides were incubated at 37°C for 30 min and then washed with 1× PBS.

[0086] Then, add tyramide modified with the fluorescent group AF 488 (TSA-AF 488) and drop it onto the glass slide (1 cm 2 Add 40 μl of the solution dropwise. Incubate the slides at 37°C for 30 minutes, then wash with 1× PBS. After washing, mount the slides and observe under a fluorescence microscope.

[0087] 1.3.3 Imaging based on biotinylated dCas9-Avi Tag

[0088] For samples using biotinylated dCas9-Avi Tag for in situ nucleic acid detection, SA-HRP can be incubated after washing in step 1.2:

[0089] Add commercially available SA-HRP and drop it onto the glass slide (1 cm 2 40 μl was added dropwise). The slides were incubated at 37°C for 30 min and then washed with 1× PBS.

[0090] Then, add tyramide modified with the fluorescent group AF 488 (TSA-AF 488) and drop it onto the glass slide (1 cm 2 Add 40 μl of the solution dropwise. Incubate the slides at 37°C for 30 minutes, then wash with 1× PBS. After washing, mount the slides and observe under a fluorescence microscope.

[0091] The results of fluorescence microscopy observations were as follows Figure 4 shown.

[0092] from Figure 4 Visible in:

[0093] (1) The in situ detection system based on the fusion protein dCas9-Avidin successfully achieved ordinary fluorescence microscopy detection, and the fluorescence signal effect was the best;

[0094] (2) The in situ detection system based on the fusion protein dCas9-Avi Tag successfully achieved ordinary fluorescence microscopy detection, but the background interference was slightly larger;

[0095] (3) The in situ detection system based on the fusion protein dCas9-HRP successfully achieved ordinary fluorescence microscopy detection, but the expression level was relatively low. The cost of raw materials is relatively high to obtain a comparable expression level.

[0096] Therefore, it can be seen that the in situ detection system based on the fusion protein dCas9-Avidin, 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 all successfully achieved ordinary fluorescence microscopy detection.

[0097] Since existing DNA in situ imaging technologies based on CRIPSR-related systems have some problems, such as complicated processes, high non-specificity, and low catalytic efficiency, which limit their application, the present invention proposes a new solution, namely, using fusion proteins and TSA technology for in situ detection. On the one hand, fusion proteins do not require covalent modification. Although constructing fusion proteins is a conventional method, different functional activities can be given to fusion proteins depending on the added domains. The present invention specifically selects suitable domains and constructs corresponding fusion proteins. Compared with covalent modification, the scheme of constructing fusion proteins is adopted in the present invention, the purification step is simple, the background interference is low, and the specificity of the probe is improved.

[0098] On the other hand, effective signal amplification is the key to achieving accurate nucleic acid detection. Tyramide 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 the results through ordinary fluorescence microscopes without having to rely on expensive confocal microscopes. In addition, the binding of signals in the TSA reaction relies on the covalent bond generated by tyramide under the peroxidase reaction, so it can withstand washing with high-intensity cleaning solutions, which effectively reduces the interference of background fluorescence. The present invention successfully combines tyramide signal amplification (TSA) technology with fusion protein technology to realize ordinary fluorescence microscopy detection of in situ detection systems.

[0099] The in situ detection system based on CRISPR / dCas fusion protein and TSA provided by the present invention is an emerging technology that effectively combines in situ detection based on CRISPR / dCas fusion protein and TSA, and has high specificity and the ability to quickly bind to the target.

[0100] The present invention also offers other advantages. For example, it can be used to detect targets bound to chromosomal DNA and histones, a feat not possible with other methods. Furthermore, the probes used in the present invention are relatively low-cost because they utilize fusion proteins, rather than modified long-chain sgRNAs or covalently modified probes.

[0101] This invention provides a CRISPR / dCas9-based nucleic acid mutation detection system for pathological sections. To ensure the specificity of the system's detection, the designed sgRNA was pre-sequenced against the NCBI nucleic acid database to confirm that it had no high-level homology matches with genomes including humans, animals, plants, and microorganisms.

[0102] The present invention involves in situ detection technology for nucleic acids in pathological sections, and its detection results can be interpreted under an ordinary 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 areas and hospitals with underdeveloped medical care.

[0103] The present invention provides a method for in situ detection of nucleic acids on pathological sections, which can simultaneously obtain tissue morphology and nucleic acid expression information, thereby improving the accuracy of pathological diagnosis.

[0104] The in situ detection method for pathological sections involved in the present invention can be regarded as a universal detection tool, which is suitable for the detection of any nucleic acid site that meets the recognition requirements of the CRISPR / dCas9 protein. When applied to different nucleic acid detection, only the sgRNA needs to be replaced.

[0105] The solution of the present invention is an effective in situ detection system based on CRISPR / dCas fusion protein and TSA, which has many advantages. The inventors believe that this technology will be widely used in the future and provide strong support for research in related fields.

[0106] Under the guidance of the present invention and the above-mentioned embodiments, it is easy for those skilled in the art to foresee that the raw materials or their equivalent substitutes, the processing methods or their equivalent substitutes listed or exemplified in the present invention can realize the present invention, and the upper and lower limit values ​​and interval values ​​of the parameters of the raw materials and processing methods can realize the present invention. The embodiments 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: It includes a fusion protein dCas9-Avidin based on the CRISPR / dCas system, biotinylated HRP, and a fluorescent group-modified tyramide solution; the dCas9-Avidin sequence is shown in SEQ ID NO: 2; In addition, the nucleic acid in situ detection system based on the fusion protein dCas9-Avidin and TSA signal amplification is detected by the following method: the fusion protein dCas9-Avidin is incubated with the target sgRNA to assemble into a dCas9-Avidin / sgRNA binary complex; the sample to be tested is contacted with the dCas9-Avidin / sgRNA binary complex, incubated and then washed; biotinylated HRP is further added, and under the specific binding of avidin-biotin, HRP binds to the vicinity of the fusion protein; fluorescent group-modified tyramide (TSA) is further added, and the fluorescent group is covalently bound to the vicinity of the target gene under the catalysis of HRP.

2. The nucleic acid in situ detection system based on CRISPR / dCas fusion protein and TSA signal amplification according to claim 1, characterized in that The dCas9-Avidin or its functional variant is encoded by the nucleic acid molecule sequence shown in SEQ ID NO:

5.

3. The nucleic acid in situ detection system based on CRISPR / dCas fusion protein and TSA signal amplification according to claim 1, characterized in that The preparation of the CRISPR / dCas system-based fusion protein is as follows: first, the sequences of the dCas9 protein and the Avidin protein are used to construct a dCas9-Avidin plasmid; then, Escherichia coli is induced to express the fusion protein; finally, the expressed fusion protein is purified.

4. The nucleic acid in situ detection system based on CRISPR / dCas fusion protein and TSA signal amplification according to claim 1, characterized in that For dCas9-Avidin, it is first combined with biotin-HRP, and the combined HRP catalyzes the fluorescent molecule-labeled TSA to perform signal amplification and color development.

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

6. Use of the nucleic acid in situ detection system based on CRISPR / dCas fusion protein and TSA signal amplification according to any one of claims 1 to 5 in the preparation of an in situ genetic detection product for pathological samples.

7. A fusion protein, characterized in that It is a 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 according to any one of claims 1 to 5.

Images