A live cell nucleic acid imaging system, method and use based on crispr and nanobodies

Abstract

The application belongs to the technical field of live cell imaging, and discloses a live cell nucleic acid imaging system, method and use based on CRISPR and nanobody, which comprises: a modified dCas9 protein expression vector containing n ALFA polypeptide tags; a modified sgRNA expression vector comprising a specific guide sequence; and a fluorescent nanobody expression vector containing a bivalent or trivalent fluorescent nanobody coding sequence, which are operatively connected, wherein a fluorescent protein is inserted between nanobody split sites as an internal tag, can be combined with the ALFA polypeptide tag carried by the dCas9 protein and stably emit light, and the free state fluorescent nanobody is self-degraded and does not emit light. The application can visualize single copy nucleic acid sequences with single molecule resolution and high signal-to-noise ratio, especially the imaging marking of single copy nucleic acid sequences in live cells.

Description

A live-cell nucleic acid imaging system, method, and application based on CRISPR and nanobodies. Technical Field

[0001] This invention relates to the field of live-cell imaging technology, and in particular to a live-cell nucleic acid imaging system, method, and application based on CRISPR and nanobodies. Background Technology

[0002] DNA, as a vital biological macromolecule, plays a crucial role in numerous life activities. Approximately 40-50% of the human genome consists of protein-coding genes, gene regulatory regions, and key non-coding regions. These regions are mostly non-repetitive sequences that control core life processes. Single-molecule resolution live-cell nucleic acid imaging is an important tool for monitoring the spatiotemporal distribution of nucleic acid molecules within cells and exploring changes in chromatin dynamics. It is of great significance for a deeper understanding of cellular function, revealing pathogenic mechanisms, and developing precision treatment strategies for diseases.

[0003] Traditional fluorescence in situ hybridization (FISH) and Oligo-FISH techniques utilize fluorescently labeled probes to specifically hybridize with denatured target DNA within cells. This method requires designing numerous fluorescent probes to target the target site and its multiple adjacent sequences, and necessitates cell fixation and DNA denaturation. It only yields a snapshot of the cellular DNA state at a given moment, failing to enable dynamic tracking of DNA sites. Furthermore, imaging techniques that directly and specifically target the target sequence or nearby exogenous sequences using fluorescent proteins present significant challenges in establishing such methods and suffer from low imaging efficiency.

[0004] After CRISPR / dCas9 protein binds to sgRNA, it can specifically target and stably bind to DNA sequences complementary to sgRNA. Through gene recombination, transfection, and other techniques, the CRISPR / dCas9-sgRNA complex can carry fluorescence and bind to DNA, enabling live-cell DNA imaging with minimal disturbance to gene function and real-time tracking of DNA state. For example, Chen Baohui et al. first fused dCas9 with EGFP, achieving live-cell imaging of telomeres under the guidance of sgRNA targeting telomere repeat sequences. However, the fluorescence intensity of a single dCas9-EGFP is very limited; this technique can only label telomere-like repeat sequences, and free dCas9-EGFP easily deposits at the nucleolus, resulting in high background signal. For live-cell non-repetitive DNA sequences, imaging requires imaging methods with sufficient signal amplification and low background signal. Although various CRISPR / Cas9-based live-cell imaging systems have been developed, high signal-to-noise ratio imaging of non-repetitive DNA sequences remains difficult to achieve due to technological limitations.

[0005] Currently, signal amplification methods for CRISPR / Cas9-based live-cell DNA non-repetitive sequence imaging systems can be mainly divided into two categories:

[0006] The first type is the non-cascade signal amplification system, which modifies the CRISPR / dCas9-sgRNA complex, such as by tandemly attaching multiple RNA aptamers (e.g., MS2, PP7, boxB) to the sgRNA or tandemly attaching a polypeptide tag (e.g., GCN4) to the dCas9 protein. This allows for in-situ fluorescent labeling by binding to fluorescent signal-carrying components (e.g., MCP, PP7, λN22, scFv). However, the number of fluorescent signal groups recruited by these systems is limited, depending on the number of RNA aptamers fused to the sgRNA and dCas9 proteins and the copy number of GCN4. For example, existing MS2-sgRNA-based imaging methods recruit a maximum of 16 MCP fluorescent proteins, while the SunTag system based on dCas9-GCN4 can recruit 24. Using a single sgRNA can only image low-degree tandem repeat sequences, and the sensitivity is still insufficient for imaging non-repetitive sequences. Furthermore, the presence of free fluorescent groups leads to high background signals. While removing the nuclear translocation signal of scFv-GFP or translocating free scFv-GFP to the cytoplasm via photoinduction can suppress background signals to some extent, the improvement in signal-to-noise ratio (SNR) is very limited (1.6-fold and 2-4-fold, respectively). Inducing rapid degradation of imaging elements is another method to improve SNR. For example, the fCRISPR system utilizes the specific binding of tDeg carrying fluorescent proteins to sgRNA-Pepper for imaging. Free tDeg undergoes rapid self-degradation, significantly improving the SNR (26-fold), and imaging of non-repetitive sequences can be achieved by tandem tDeg with 11 copies of GFP. However, because this system has limited ability to recruit fluorescent signals to target sites—that is, when imaging components are overexpressed in cells, they can self-bind to form stable free fluorescent dCas9 / Pepper-sgRNA / tDeg-FP complexes—background fluorescence still occurs.

[0007] The second type is the cascade signal amplification system, which, based on the first type, introduces bridging components to form a superposition of multiple signal groups. For example, the CRISPR FISHer system constructs a foldon-GFP-PCP trimer protein, using the CRISPR / dCas9-sgRNA (carrying the PP7 motif)-target DNA complex as a fulcrum. Through PCP-PP7 interaction, it achieves the cascade assembly of sgRNA-PP7 and foldon-GFP-PCP, enabling imaging of non-repetitive sequences through high signal amplification. However, this system is limited by the natural polymerization characteristics of foldon, making multicolor imaging difficult, and the modified sgRNA backbone is prone to aggregation, leading to significant non-specific signals and failing to solve the problem of high background signal. The SIMBA system consists of a target binding arm composed of dCas9-GCN4 / scFv-FKBP and a signal amplification arm composed of FRB-mCherry-HP1α. FKBP binds to FRB under rapamycin induction and forms a signal amplification complex at the target site via HP1α-mediated multivalent interactions, consisting of a target-binding arm and a signal amplification arm, thereby visualizing non-repetitive sequences. However, HP1α is a natural component of human cells and readily binds nonspecifically to chromatin, leading to false positive signals.

[0008] In summary, developing a live-cell imaging system with high signal-to-noise ratio and capable of labeling non-repetitive DNA sequences is a key problem that needs to be solved. Realizing live-cell DNA non-repetitive sequence imaging is of great significance and application value for fully revealing gene expression regulation, cell function, tissue development, and disease mechanisms in life sciences and medical sciences. Summary of the Invention

[0009] This invention provides a live-cell nucleic acid imaging system, method, and application based on CRISPR and nanobodies to address the aforementioned technical problems in the prior art.

[0010] To provide a basic understanding of some aspects of the disclosed embodiments, a brief summary is given below. This summary is not intended as a general commentary, nor is it intended to identify key / important components or to describe the scope of protection of these embodiments. Its sole purpose is to present some concepts in a simple form as a prelude to the detailed description that follows.

[0011] The purpose of this invention is to improve the signal-to-noise ratio of an imaging system and achieve low-background imaging of non-repetitive DNA sequences. It utilizes a CRISPR / dCas9-sgRNA complex to target live-cell nucleic acid sequences and achieves high signal-to-noise ratio imaging through a cascaded fluorescence signal amplification system mediated by antigen-dependent luminescence fluorescent nanobodies, abbreviated as the BRIGHT system. B i-NI R-Fb-mediated cascade-dependent I illumination for non-repetitive G enomic loci H igh-SNR T This imaging system can significantly improve the signal-to-noise ratio of imaging, greatly reducing background noise while amplifying the signal, thereby achieving high signal-to-noise ratio imaging of non-repetitive DNA sequences in live cells.

[0012] According to a first aspect of the present invention, a live-cell nucleic acid imaging system based on CRISPR and nanobodies is provided.

[0013] In one embodiment, the CRISPR- and nanobody-based live-cell nucleic acid imaging system includes:

[0014] The modified dCas9 protein expression vector comprises dCas9 and n tandem ALFA polypeptide tags (n=1,4,8,16,24), which are operably linked together and the linking method is not fixed, and the optimal linking method can be selected according to actual needs.

[0015] The modified sgRNA expression vector comprises an sgRNA backbone and a specific guide sequence for the target sequence. Fluorescent sequences such as the mcherry sequence inherent in the plasmid need to be removed to avoid interfering with the interpretation of imaging results.

[0016] A fluorescent nanobody expression vector comprising: a heavy chain variable region sequence derived from pure heavy chain antibodies from camels and sharks, wherein a fluorescent protein sequence is inserted at the N65 / C66 site, capable of recognizing a tandem ALFA polypeptide tag in a modified dCas9 protein expression vector. This expression vector contains bivalent or trivalent fluorescent nanobody encoding sequences; wherein the fluorescent protein sequence can be a multi-color fluorescent protein sequence spanning the entire spectrum from ultraviolet to near-infrared.

[0017] In one embodiment, the modified dCas9 protein expression vector, the modified sgRNA expression vector, and the fluorescent nanobody expression vector were transfected into a cell line.

[0018] In one embodiment, the dCas9 sequence is as shown in SEQ ID No:1.

[0019] In one embodiment, the dCas9 protein is derived from Staphylococcus aureus. Those skilled in the art can replace it with dCas9 protein derived from, but not limited to, Neisseria meningitidis, as needed.

[0020] In one embodiment, the antigenic polypeptide tag fused to the dCas9 sequence is an ALFA polypeptide, the ALFA sequence of which is shown in SEQ ID No:2.

[0021] n×ALFA represents n ALFA peptides linked in tandem, which can be linked by adapter sequences selected from commonly used adapter sequences in the field.

[0022] In one embodiment, in the BRIGHT system, the number of dCas9 tandem ALFAs is 1, 4, 8, 16, and 24, and the dCas9-n×ALFA (n=1, 4, 8, 16, 24) fusion protein sequences are shown in SEQ ID No: 3, 4, 5, 6, and 7, respectively.

[0023] In one embodiment, in the BRIGHT system, the fluorescent nanobody is an ALFA nanobody with an inserted fluorescent protein of miRFP670nano3 near-infrared fluorescent protein. The fluorescent nanobody has self-degradation properties, that is, in the absence of dCas9-n×ALFA, the antibody is degraded through the ubiquitin-proteasome pathway. Only when dCas9-n×ALFA is expressed and stably bound to the fluorescent nanobody can it emit stable red fluorescence.

[0024] In one specific implementation, the fluorescent nanobody expression vector contains m fluorescent nanobody coding sequences, where m is two (bivalent fluorescent nanobody, Bi-NIR-Fb). ALFA ) or 3 (trivalent fluorescent nanobodies, Tri-NIR-Fb ALFA The Bi-NIR-Fb ALFA The sequence is shown in SEQ ID No:8, the Tri-NIR-Fb ALFA The sequence is shown in SEQ ID No:9.

[0025] In other embodiments, the near-infrared fluorescent protein in the fluorescent nanobody can be replaced by, but is not limited to, green fluorescent protein, red fluorescent protein, and blue fluorescent protein. The screening principles are as follows: (1) It can be stably expressed in mammalian cells; (2) It does not require exogenous cofactors to avoid potential effects on cell function; (3) It is small in size (less than 20 kDa), exists in monomeric form, and is suitable as an internal tag inserted into Bi-Fb. ALFA (4) The structure is compact, with the N-terminus and C-terminus close to each other in space, ensuring no interference with Bi-Fb. ALFA Spatial conformational changes during antigen recognition and binding. Equivalent imaging effects can be achieved using fluorescent proteins of other colors, such as inserting miniGFP1 into Bi-Fb.ALFA Bi-miniGFP-Fb was constructed at the N65 / C66 sites. ALFA The green fluorescent nanobody has the sequence shown in SEQ ID No: 10.

[0026] The expression of fluorescent nanobody is driven by the CMV promoter, the EF-1α core promoter, the hPGK promoter, and the miniCMV promoter, the promoter sequences of which are shown in SEQ ID No: 11, 12, 13, and 14, respectively. Preferably, the hPGK promoter is the best for imaging effect.

[0027] For dCas9 recombinant protein, conventional promoters such as the SV40 promoter and CMV promoter can be used to continuously initiate the expression of dCas9 recombinant protein and sgRNA. Those skilled in the art can choose appropriate promoters.

[0028] For sgRNA, an appropriate promoter such as the U6 promoter can be selected.

[0029] Depending on the needs of the practical application, those skilled in the art can easily select appropriate plasmids to construct the above-mentioned expression vectors (i.e., modified dCas9 protein expression vectors, modified sgRNA expression vectors, and fluorescent nanobody expression vectors). Available plasmids include, but are not limited to, pcDNA3.1, PX330, and lentiviruses.

[0030] In the imaging labeling system based on CRISPR and fluorescent nanobodies of the present invention, the dCas9-n×ALFA expression vector can be replaced by dCas9-n×ALFA protein or a cell line that stably expresses dCas9-n×ALFA protein.

[0031] The live-cell nucleic acid imaging system based on CRISPR and nanobodies of this invention can achieve high signal-to-noise ratio imaging labeling of non-repetitive sequences. This is achieved by the formation of an exponential cascade signal amplification complex of dCas9 recombinant protein and fluorescent nanobodies at the target nucleic acid site, while the free fluorescent nanobodies undergo self-degradation. This is achieved using dCas9-16×ALFA and Bi-NIR-Fb... ALFA For example, the process of imaging marker aggregation is illustrated as follows: (1) Cascade process: dCas9-16×ALFA first forms a complex with sgRNA and binds to the DNA site targeted by sgRNA. Then, dCas9-16×ALFA recruits Bi-NIR-Fb through antigen-antibody interaction. ALFA Bi-NIR-Fb ALFAThe dual antigen-binding arm can bind to two antigenic epitopes simultaneously, thus acting as a "bridge" to bind to the second layer of dCas9-16×ALFA, which in turn recruits more Bi-NIR-Fb. ALFA Therefore, the BRIGHT system of the present invention utilizes dCas9-16×ALFA and Bi-NIR-Fb ALFA The repeated recruitment and combination ultimately form dCas9-n×ALFA / Bi-NIR-Fb ALFA The multi-layered cascade complex. This aggregate contains multiple near-infrared fluorescent protein groups, thereby achieving high-magnification of the fluorescence signal. (2) Background denoising: Bi-NIR-Fb ALFA It can bind to two molecules of ALFA, only Bi-NIR-Fb ALFA Red fluorescence is emitted only when both antigen-binding arms are stably bound to dCas9-16×ALFA. In the free state or when a single antigen-binding arm is stably bound to dCas9-16×ALFA, it will be degraded through the ubiquitin-proteasome pathway and thus cannot emit light. (3) Imaging effect: Signal cascading causes multiple near-infrared fluorescent proteins to aggregate at the target site, which greatly improves the fluorescence intensity of the signal point in the BRIGHT system. Background denoising causes the free near-infrared fluorescent proteins to self-degrade, which greatly reduces the background fluorescence signal intensity, thus achieving the effect of high signal-to-noise ratio imaging of non-repetitive sequences using only one sgRNA (Figure 1).

[0032] According to a second aspect of the present invention, a live-cell nucleic acid imaging method based on CRISPR and nanobodies is provided.

[0033] In one embodiment, the CRISPR- and nanobody-based live-cell nucleic acid imaging method includes:

[0034] A live-cell nucleic acid imaging system based on CRISPR and nanobodies was constructed, including a modified dCas9 protein expression vector, a modified sgRNA expression vector, and a fluorescent nanobodies expression vector.

[0035] The components of the constructed CRISPR- and nanobody-based live-cell nucleic acid imaging system were transfected into the cells to be tested.

[0036] Fluorescent signal points formed by a live-cell nucleic acid imaging system based on CRISPR and nanobodies were observed using live-cell imaging instruments such as confocal microscopy and multimodal super-resolution microscopy.

[0037] Cell transfection is a conventional method that introduces exogenous DNA sequences into cells. It includes transfection using lentiviruses, transfection reagents such as jetPRIME® transfection reagent (Polyplus) and Lipofectamine 3000 transfection reagent (Thermo FISHer), and electroporation.

[0038] The signal points generated by the BRIGHT system in this invention have sufficient fluorescence signal intensity, and can be observed and photographed using a confocal microscope or a regular optical microscope.

[0039] In one embodiment, the BRIGHT live-cell DNA imaging system was constructed by targeting the telomeres of HEK293T cells, and its telomere imaging performance was compared with that of the CRISPR SunTag system (hereinafter referred to as the SunTag system) developed by Ronald D.Val and the dCas9 / 2×PP7-sgRNA / PCP-3×GFP imaging system (hereinafter referred to as the PP7-PCP system) developed by Ma Hanhui in CRISPRainbow.

[0040] In one embodiment, the selected target DNA sequence includes various types such as high-copy highly tandem repeat sequences, low-copy low-copy low-copy tandem repeat sequences, and single-copy non-repetitive sequences, which may be located on or outside chromosomal DNA, and may be any sequence derived from the human genome or introduced from abroad.

[0041] In one embodiment, the imaging specificity of the BRIGHT live-cell DNA non-repetitive sequence imaging system was verified and the preferred conditions for non-repetitive sequence visualization were determined. The specificity of single-copy non-repetitive DNA sequence imaging was verified by co-localization with Oligo FISH imaging technology. Preferably, dCas9-16×ALFA / Bi-NIR-Fb was used. ALFA and dCas9-24×ALFA / Bi-NIR-Fb ALFA Visualizing non-repetitive sequences yields the highest signal-to-noise ratio.

[0042] In one embodiment, the selected labeled cell lines are HeLa, HepG2, and U2OS cell lines. Those skilled in the art can select appropriate cell lines for imaging as needed, including but not limited to various mammalian cell lines.

[0043] In one embodiment, the BRIGHT system of the present invention can be used for labeling extrachromosomal DNA sequences (such as eccDNA, HBV cccDNA, etc.).

[0044] According to a third aspect of the present invention, a CRISPR-based and nanobody-based live-cell nucleic acid imaging system is provided for use in the preparation of reagent kits.

[0045] The technical solutions provided by the embodiments of the present invention may include the following beneficial effects:

[0046] (1) This invention establishes a novel method for live-cell nucleic acid imaging labeling based on CRISPR and fluorescent nanobodies, providing a technical foundation for live-cell nucleic acid sequence imaging and enabling high signal-to-noise ratio imaging of single-copy sites. The technical approach provided by this invention can offer empirical methods for the development of new live-cell nucleic acid imaging technologies.

[0047] (2) The live cell nucleic acid imaging system proposed in this invention has simple components and only requires antigen-antibody interaction between dCas9-tandem ALFA polypeptide fusion protein and fluorescent nanobody to achieve cascade signal amplification.

[0048] (3) The live cell nucleic acid imaging system proposed in this invention is based on the multivalent binding ability, luminescence characteristics and antigen-dependent stability of fluorescent nanobodies. It simultaneously realizes cascade signal amplification based on antigen-antibody interaction and background noise reduction based on the self-degradation of free fluorescent elements, thereby maximizing the high signal-to-noise ratio imaging of DNA non-repetitive sequences and significantly improving the imaging effect.

[0049] (4) The live cell nucleic acid imaging system proposed in this invention is operable. By tandemly connecting different copies of fluorescent nanobodies, bivalent and trivalent nanobodies can be constructed. By enabling nanobodies to carry different fluorescent proteins, live cell imaging systems with different fluorescent labels can be flexibly designed, thereby achieving multicolor imaging.

[0050] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and are not intended to limit the invention. Attached Figure Description

[0051] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0052] Figure 1 is a schematic diagram illustrating the construction of fluorescent nanobodies and the BRIGHT live-cell imaging system according to an exemplary embodiment; by inserting miRFP670nano3 near-infrared fluorescent protein into ALFA nanobodies (Nb ALFA Near-infrared fluorescent nanobodies (NIR-Fb) with antigen-dependent stability were constructed from the N65 / C66 sites. ALFA Using (G4S)5 connectors to connect two NIR-Fb ALFALinked to form bivalent infrared fluorescent nanobodies (Bi-NIR-Fb). ALFA dCas9-16×ALFA recruits Bi-NIR-Fb via antigen-antibody interaction. ALFA Bi-NIR-Fb ALFA The dual antigen-binding arm can bind to two antigenic epitopes simultaneously, thus acting as a "bridge" to ultimately form dCas9-n×ALFA / Bi-NIR-Fb. ALFA It is a multi-layered cascade complex. However, when it is in a free state or when a single antigen-binding arm is stably bound to dCas9-16×ALFA, it will be degraded via the ubiquitin-proteasome pathway, thus failing to emit light;

[0053] Figure 2 is a schematic diagram illustrating the construction and validation results of dCas9-n×ALFA (n=1, 4, 8, 16, 24) fusion protein expression plasmids according to an exemplary embodiment; Figure 2A is a schematic diagram of constructing dCas9-n×ALFA fusion protein expression plasmids based on dCas9-24×GCN4 fusion protein expression plasmids; Figure 2B is a diagram of the enzyme digestion and electrophoresis results of dCas9-n×ALFA plasmids; Figure 2C is a diagram of the telomere labeling results of dCas9 and dCas9-n×ALFA.

[0054] Figure 3 illustrates a Bi-NIR-Fb according to an exemplary embodiment. ALFA Schematic diagram of expression plasmid construction and validation; Figure 3A shows the mRNA expression levels of dCas9-24×ALFA and dCas9-24×GCN4 in each group detected by RT-qPCR; Figure 3B shows the Bi-NIR-Fb expression levels in each group detected by RT-qPCR. ALFA Figure 3C shows the mRNA expression level of the ALFA peptide group, GCN4 peptide group, and proteasome inhibitor group after 24 h of transfection; Figure 3D shows the fluorescence intensity of TagBFP in each of the above groups; Figure 3E shows the near-infrared fluorescence intensity of each of the above groups.

[0055] Figure 4 illustrates Bi-miniGFP-Fb according to an exemplary embodiment. ALFA Construction diagram of green fluorescent nanobody; Figure 4A shows the protein structure and sequence composition of miniGFP1; Figure 4B shows Bi-miniGFP-Fb ALFA Protein structure prediction results;

[0056] Figure 5 illustrates a Bi-NIR-Fb according to an exemplary embodiment. ALFA Figure 5A shows the mRNA expression levels of dCas9-24×ALFA in different promoter groups detected by RT-qPCR; Figure 5B shows the mRNA expression levels of Bi-NIR-Fb in different promoter groups detected by RT-qPCR.ALFA mRNA expression levels; Figure 5C shows the expression levels of Bi-NIR-Fb under different promoter groups. ALFA Comparison of mRNA expression levels; Figure 5D shows Bi-NIR-Fb carrying different promoters. ALFA Microscopic results after co-transfection with dCas9-24×ALFA plasmid; Figure 5E shows the results of Bi-NIR-Fb co-transfection with different promoter groups. ALFA The results of the comparison of near-infrared fluorescence intensity;

[0057] Figure 6 is a diagram illustrating telomere imaging results using the BRIGHT system, SunTag system, and PP7-PCP system according to an exemplary embodiment. The first row of Figure 6A shows the maximum intensity projection of the Z-axis stack of representative telomere marking results from the BRIGHT, SunTag, and PP7-PCP systems. The second row is an XYZ three-dimensional image of the representative results from the first row. The third row shows the fluorescence intensity of representative signal points obtained through Plot Profile analysis of selected signal points in the first row image as a function of the spatial distance of the lines. Figure 6B is a statistical result of the fluorescence intensity of telomere imaging signal points from the three systems. Figure 6C is a statistical result of the signal-to-noise ratio of telomere imaging from the three systems.

[0058] Figure 7 is a microscopic image showing the results of using the BRIGHT system to label highly tandemly repetitive sequences of the Chr3, Chr13 and MUC4 genes according to an exemplary embodiment;

[0059] Figure 8 is a microscopic image of low-degree tandem repeat sequences on Chr9, Chr13, Chr17, Chr19 and Chr21 labeled using the BRIGHT system according to an exemplary embodiment.

[0060] Figure 9 is a diagram illustrating the labeling results of non-repetitive sequences according to an exemplary embodiment; Figure 9A shows the microscopic results of labeling MUC4.1, MUC4.2, MUC4.3, MUC4.4, PPP1R2, and SOX1 non-repetitive sequences using the BRIGHT system; Figure 9B shows the results of labeling using Bi-miniGFP-Fb. ALFA Microscopic image of the MUC4.1 site;

[0061] Figure 10 is an experimental result of co-localization of the BRIGHT system marker MUC4.1 and the Oligo-FISH marker MUC4Rep according to an exemplary embodiment;

[0062] Figure 11 illustrates the use of dCas9-n×ALFA (n=1, 4, 8, 16, 24), Bi-NIR-Fb according to an exemplary embodiment. ALFA Bivalent nanobodies and Tri-NIR-FbALFA Figure 11A shows the results of labeling MUC4.1 sites with trivalent nanobodies; Figure 11A shows the results under a microscope and the curves of fluorescence intensity of representative signal points as a function of spatial distance of the lines obtained by Plot Profile analysis; Figure 11B shows the statistical results of the diameter of signal points labeled with MUC4.1 sites in each group; Figure 11C shows the statistical results of the signal-to-noise ratio of labeled MUC4.1 sites in each group.

[0063] Figure 12 is a microscopic image of the MUC4.1 nontandem repeat sequence labeled using the BRIGHT system in HeLa, HepG2 and U2OS cells according to an exemplary embodiment. Detailed Implementation

[0064] The following description and accompanying drawings fully illustrate specific embodiments described herein to enable those skilled in the art to practice them. Some portions and features of certain embodiments may be included in or replace portions and features of other embodiments. The scope of the embodiments herein includes the entire scope of the claims, as well as all available equivalents of the claims.

[0065] In this document, unless otherwise stated, the term "multiple" means two or more.

[0066] In this article, the character " / " indicates that the objects before and after it are in an "or" relationship. For example, A / B means: A or B.

[0067] In this article, the term "and / or" describes an association between objects, indicating that three relationships can exist. For example, A and / or B means: A or B, or A and B.

[0068] Where there is no conflict, the embodiments and features in the embodiments of the present invention can be combined with each other.

[0069] Example 1: Preparation and Validation of Components of the BRIGHT Live-Cell DNA Imaging System

[0070] This embodiment describes the plasmids used in other embodiments of the present invention, and the specific sequence information of these plasmids is detailed in the sequence listing.

[0071] (1) Construction and validation of dCas9-n×ALFA fusion protein expression plasmid

[0072] ① Construction of dCas9-n×ALFA fusion protein expression plasmid: Based on the dCas9-24×GCN4 fusion protein expression plasmid (Addgene #60910) in the SunTag system, the 24×GCN4 sequence was removed by double digestion with BamHI and SalI. Different copies (1, 4, 8, 16, 24) of the synthesized gene were tandemly inserted into the digested vector to construct the dCas9-n×ALFA fusion protein expression plasmid (Figure 2A). This vector drives the expression of the dCas9-n×ALFA fusion protein under the regulation of the SV40 promoter, and the expressed fusion protein can target and recognize specific sites under the guidance of sgRNA. The dCas9-1×ALFA, dCas9-4×ALFA, dCas9-8×ALFA, dCas9-16×ALFA, and dCas9-24×ALFA fusion protein sequences provided in this invention are shown in SEQ ID No: 3, 4, 5, 6, and 7, respectively.

[0073] ② Validate the target binding ability of the dCas9-n×ALFA fusion protein: Validate the target binding ability of the dCas9-n×ALFA fusion protein based on the PP7-PCP system.

[0074] The experimental steps are as follows:

[0075] a. HEK293T cells were plated and cultured overnight. Transfection was performed after the cell density reached 70%.

[0076] b. 30 minutes before transfection, replace the culture medium with 2 mL of Opti-MEM serum-reduced medium (phenol red-free, ThermoFISHer).

[0077] c. Add 200µL of jetPRIME® buffer (Polyplus) to a 1.5 mL EP tube.

[0078] d. Add each plasmid to 200µL jetPRIME® buffer according to the plasmid transfection dosage in Table 1. Vortex for 10s and then detach.

[0079] e. Vortex the jetPRIME® transfection reagent for 5 seconds and then detach it momentarily.

[0080] f. Add the corresponding volume of jetPRIME® transfection reagent according to the ratio of total nucleic acid mass (µg): jetPRIME® transfection reagent (µL) = 1:2, vortex for 1 second and then instantly separate.

[0081] g. Incubate at room temperature for 10 min.

[0082] h. Slowly add the transfection mixture to the culture dish, gently shake to mix, and incubate at 37°C for 4-6 hours.

[0083] i. After 4-6 hours, replace the transfection medium with complete culture medium and return the plate to the incubator.

[0084] j. The results were observed using a confocal fluorescence microscope 24 hours after transfection.

[0085] Table 1 Transfection System

[0086] ;

[0087] Experimental results:

[0088] Figure 2B shows the electrophoretic verification results of dCas9-n×ALFA (n=1, 4, 8, 16, 24) digested with BamHI and SalI. Figure 2C shows the telomere labeling results of the dCas9 positive control group and the dCas9-n×ALFA experimental group. Similar to the positive control group, the dCas9-n×ALFA fusion protein was able to target telomeres under the guidance of 2×PP7-sgRNA, indicating that the fusion of the tandem ALFA peptide does not affect the dCas9 target binding ability.

[0089] (2) Construction and validation of fluorescent nanobody expression plasmids

[0090] ① Constructing Bi-NIR-Fb ALFA Fusion protein expression plasmid: Bi-NIR-Fb was obtained through gene synthesis. ALFA The bivalent nanobody encoding sequence, in which the two nanobody sequences are linked by a (G4S)5 linker peptide (Figure 1), was inserted between the KpnI and BamHI restriction sites of the CMV-pcDNA3.1(+) plasmid vector to construct Bi-NIR-Fb. ALFA A fusion protein expression plasmid. This vector drives Bi-NIR-Fb expression under the regulation of the CMV promoter. ALFA Expression of the fusion protein, namely Bi-NIR-Fb ALFA The sequence is shown in SEQ ID No:8.

[0091] ②Verification of Bi-NIR-Fb ALFA Antigen-dependent stability of fusion proteins: setting up dCas9-24×ALFA and Bi-NIR-Fb ALFA Co-transfection (ALFA peptide group), dCas9-24×GCN4 and Bi-NIR-Fb ALFACo-transfection (GCN4 peptide group) and dCas9-24×GCN4 with Bi-NIR-Fb ALFA Co-transfection for 16 h was followed by treatment with the proteasome inhibitor MG132 for 6 h (proteasome inhibition group). Transfection was performed according to Table 2. 24 h post-transfection, the concentrations of dCas9-24×ALFA, dCas9-24×GCN4, and Bi-NIR-Fb in each group were analyzed by dye-based RT-qPCR and confocal microscopy. ALFA The mRNA and protein expression levels were measured. β-catenin was used as an internal control in the dye-based RT-qPCR method. The primer sequences used in the dye-based RT-qPCR method are shown in Table 3.

[0092] Table 2 Co-transfection system

[0093] ;

[0094] Table 3 Primer sequences used in dye-based RT-qPCR experiments

[0095] ;

[0096] Note: RT-qPCR detects the dCas9 fragments of dCas9-24×ALFA and dCas9-24×GCN4, therefore the primers before and after dCas9-24×ALFA and dCas9-24×GCN4 are the same.

[0097] Experimental results:

[0098] Figure 3A shows that the mRNA expression levels of dCas9-24×ALFA and dCas9-24×GCN4 are consistent in each group. The specific Ct values ​​are shown in Table 4.

[0099] Figure 3B shows the Bi-NIR-Fb groups. ALFA The mRNA expression levels were consistent, and the specific Ct values ​​are shown in Table 4.

[0100] Figures 3C and 3D show that TagBFP blue fluorescence was visible in all groups, and there was no statistically significant difference in fluorescence intensity (p>0.05), indicating that the expression levels of dCas9-24×ALFA and dCas9-24×GCN4 fusion proteins were consistent among the groups.

[0101] Figure 3C shows that the GCN4 polypeptide group has no obvious near-infrared fluorescence, while the ALFA polypeptide group shows strong near-infrared fluorescence. Figure 3E shows that the quantitative analysis of near-infrared fluorescence intensity shows that the fluorescence intensity of the ALFA polypeptide group is 2070.28 times that of the GCN4 polypeptide group, indicating that Bi-NIR-Fb ALFAThe GCN4 peptide specifically binds to the ALFA peptide and is stable in dependence on it. When the MG132 proteasome inhibitor is added to block the proteolytic activity of the 26S proteasome complex, the near-infrared fluorescence of the GCN4 peptide group recovers to 75.53% of that of the ALFA peptide group, and the fluorescence intensity is 1563.74 times that of the GCN4 peptide group, indicating that Bi-NIR-Fb ALFA Degraded via the proteasome pathway.

[0102] Table 4. Detection results of RT-qPCR

[0103] ;

[0104] ③ Constructing Tri-NIR-Fb ALFA Fusion protein expression plasmid: Tri-NIR-Fb was obtained through gene synthesis. ALFA The trivalent nanobody encoding sequence is linked together by a (G4S)5 linker peptide. This encoding sequence was inserted between the KpnI and BamHI restriction sites of the CMV-pcDNA3.1(+) plasmid vector to construct Tri-NIR-Fb. ALFA Fusion protein expression plasmid. This vector drives Tri-NIR-Fb expression under the regulation of the CMV promoter. ALFA The fusion protein expression, namely Tri-NIR-Fb ALFA The sequence is shown in SEQ ID No:9.

[0105] ④ Expanding the imaging spectral range of the BRIGHT system: miniGFP1 is a naturally occurring monomeric green fluorescent protein found in Arabidopsis thaliana. It is 12.6 kDa in size, composed of 110 amino acids (Figure 4A), with a maximum excitation wavelength of 450 nm and a maximum emission wavelength of 499 nm. Its monomeric, small size, stable existence in mammalian cells, and lack of exogenous cofactors make it suitable for developing self-degrading Bi-Fb with other spectral ranges. ALFA The requirement is to insert the miniGFP protein into Bi-Fb. ALFA The N65 / C66 sites (Figure 4B) were used for protein structure prediction using AlphaFold3, and Bi-miniGFP-Fb was constructed. ALFA Green fluorescent nanobody (sequence corresponding to SEQ ID No: 10).

[0106] ⑤ Optimize Bi-NIR-Fb ALFA Promoter sequence: Based on Bi-NIR-Fb carrying CMV promoter (strong promoter) ALFAThe fusion protein expression plasmid was prepared by removing the CMV promoter sequence using MluI and SacI restriction enzymes. The EF-1α core promoter, the hPGK promoter (medium strength), and the miniCMV (weak promoter) sequences were then inserted into this site to construct Bi-NIR-Fb plasmids carrying promoters of varying strengths. ALFA The expression plasmids, with nucleotide sequences of each promoter corresponding to SEQ ID Nos: 11, 12, 13, and 14, respectively. They were then processed according to the instructions for using dCas9-24×ALFA and Bi-NIR-Fb. ALFA The molar mass ratio was 2:1, and dCas9-24×ALFA (0.30 pmol) and Bi-NIR-Fb carrying different promoters were co-transfected. ALFA Plasmid (0.15 pmol) was injected into HEK293T cells, and the levels of dCas9-24×ALFA and Bi-NIR-Fb in each group were detected by dye-based RT-qPCR. ALFA The mRNA level was measured (the primer sequences used in the dye-based RT-qPCR experiment are shown in Table 3, with β-catenin as an internal reference). Near-infrared fluorescence intensity was observed using confocal microscopy. Promoters with high mRNA levels and fluorescence intensity, and without aggregation or non-specific nucleolar binding, were selected as the optimal conditions.

[0107] Experimental results:

[0108] Figure 5A shows that the mRNA expression levels of dCas9-24×ALFA were consistent across groups. Specific Ct values ​​are shown in Tables 5.1 and 5.2.

[0109] Figure 5B shows Bi-NIR-Fb ALFA The mRNA expression levels were CMV promoter > EF-1α core promoter > hPGK promoter > miniCMV promoter. Specific Ct values ​​are shown in Tables 5.1 and 5.2.

[0110] Figure 5C shows the Bi-NIR-Fb regulated by the CMV promoter, EF-1α core promoter, and hPGK promoter. ALFA The mRNA expression levels were 64.00, 36.50, and 5.31 times higher than those of the miniCMV promoter, respectively.

[0111] Figure 5D shows Bi-NIR-Fb carrying different promoters. ALFAMicroscopic results after co-transfection with dCas9-24×ALFA plasmid. TagBFP blue fluorescence was used to indicate the expression of dCas9-24×ALFA protein in each group. Near-infrared fluorescence intensity showed significant differences among groups. CMV promoter-induced group showed obvious near-infrared fluorescence patch aggregation. EF-1α core promoter group showed nucleolar nonspecific binding. hPGK promoter group showed no obvious fluorescence aggregation or nucleolar nonspecific binding. miniCMV promoter showed no obvious near-infrared fluorescence.

[0112] Figure 5E shows the Bi-NIR-Fb regulated by the CMV promoter, EF-1α core promoter, and hPGK promoter. ALFA The near-infrared fluorescence intensities were 24.25, 6.39, and 3.03 times that of the miniCMV promoter, respectively.

[0113] With consistent dCas9-24×ALFA expression across groups, different promoter regulation leads to Bi-NIR-Fb ALFA The differences in expression levels are due to the differences in expression regulation caused by different promoters. Among them, the hPGK promoter can control Bi-NIR-Fb ALFA Moderate expression levels and no Bi-NIR-Fb ALFA The aggregation-induced fluorescent patches bind nonspecifically to the nucleolus, therefore hPGK was chosen as the Bi-NIR-Fb standard. ALFA The optimal promoter for a plasmid.

[0114] Table 5.1 One of the detection results of RT-qPCR

[0115] ;

[0116] Table 5.2 Detection results of RT-qPCR (Part 2)

[0117] ;

[0118] (3) Construction of sgRNA expression plasmids: Based on the plasmid (Addgene #51024) expressing telomeric sgRNA in the SunTag system, the mCherry sequence was removed by double digestion with AgeI and SgrAI. The complementary sticky ends formed after double digestion were ligated by ligase to construct the telomeric sgTelomere plasmid. sgRNA expression plasmids targeting other sites were constructed using the Gibson seamless cloning method.

[0119] Example 2: Construction of the BRIGHT live-cell DNA imaging system targeting telomeres

[0120] To perform telomere live-cell imaging, sgRNA expression plasmids targeting telomeres (experimental group) and those targeting GAL4 (negative control group, as this target site is not present in human cells) were transfected with dCas9-24×ALFA and Bi-NIR-Fb according to the transfection system in Table 6. ALFA The expression plasmid was transfected into HEK293T cells, and the labeling results were observed using confocal microscopy 24 hours later. To verify the signal amplification capability of the BRIGHT system, telomeres were also labeled in HKE293T cells using the SunTag live-cell imaging system (based on dCas9 protein modification) and the PP7-PCP imaging system (based on sgRNA modification) according to the transfection system in Table 6. The results were observed 24 hours after transfection. Using the Straight Line tool in ImageJ, lines were drawn along the central axis of representative signal points, and the fluorescence intensity versus the spatial distance of the drawn lines was plotted using the Plot profile tool. The fluorescence intensity (Signal) of 20 signal points in each group was quantitatively analyzed using ImageJ software, and the average background fluorescence intensity (Noise) was calculated by randomly selecting 30 circular regions of the same size (10×10 pixels) in the background area using the Oval Selection tool.

[0121] Table 6 Transfection System

[0122] ;

[0123] Experimental results:

[0124] Figure 6A shows that the near-infrared fluorescence signal of telomere-like structures labeled by the BRIGHT imaging system is uniformly distributed throughout the cell nucleus, with a bright signal and extremely low background. The negative control group targeting GAL4 only showed a uniformly diffuse near-infrared fluorescence background signal, with no non-specific binding observed, further demonstrating the telomere labeling specificity of the BRIGHT system. In contrast, the SunTag system exhibited significant nuclear background fluorescence due to the stable free flow of constitutively overexpressed scFv-sfGFP outside the target site. The PP7-PCP system had a signal amplification of only 6-fold, resulting in a weaker fluorescence signal.

[0125] Figures 6B and 6C show the fluorescence signal intensity and SNR analysis of 20 telomere signal points from the three labeling systems. The average signal-to-noise ratio of telomere imaging in the BRIGHT system of this invention is 72.68±13.28, which is 35.34 times and 13.22 times that of the SunTag system and the PP7-PCP system, respectively.

[0126] In summary, this invention significantly reduces background fluorescence while improving signal intensity, achieving superior telomere labeling results, thanks to Bi-NIR-Fb.ALFA and dCas9-24×ALFA-mediated exponential signal amplification and free Bi-NIR-Fb ALFA Its self-degradable properties.

[0127] Example 3: Labeling other tandem repeat sequences using the BRIGHT live-cell DNA imaging system

[0128] The CRISPRbar database (http: / / genome.ucf.edu / CRISPRbar / ) was used to screen for highly tandemly repetitive sequences (copy number > 100) located on different chromosomes (target sequences corresponding to SEQ ID Nos: 23, 24, and 25, respectively) and low-transcriptional repetitive sequences (copy number 5–100) (target sequences corresponding to SEQ ID Nos: 26, 27, 28, 29, 30, and 31, respectively). Specific sequences are shown in Table 7. These sequences are known, belong to the public domain, and have been described in other literature. sgRNA, dCas9-24×ALFA, and Bi-NIR-Fb targeting each chromosome were then used. ALFA The expression plasmid was transfected into HEK293T cells, and the labeling results were observed using confocal microscopy 24 hours later.

[0129] Table 7. sgRNA information targeting other tandem repeat sequences

[0130] ;

[0131] Experimental results:

[0132] Figures 7 and 8 show the microscopic results of labeling highly tandem repeat sequences (copy number >100, located in Chr3, Chr13 and MUC4 genes) and lowly tandem repeat sequences (copy number 5-100, located in Chr9, Chr13, Chr17, Chr19 and Chr21) on different chromosomes using the BRIGHT system, respectively. Two bright near-infrared fluorescent signal spots are visible in the cell nucleus, indicating that the new method can achieve the labeling of repeat sequences outside of telomeres.

[0133] Example 4: Labeling Non-Tandem Repeat Sequences with the BRIGHT Live-Cell DNA Imaging System

[0134] Six non-repetitive sequences were selected from chromosome 3 MUC4 (4 sequences), chromosome 3 PPP1R2 (1 sequence), and chromosome 13 SOX1 (1 sequence), with target sequences corresponding to SEQ ID Nos. 32, 33, 34, 35, 36, and 37, respectively. The specific sequences are shown in Table 8. These sequences are known, belong to the public domain, and have been described in other literature. The sgRNA, dCas9-24×ALFA, and Bi-NIR-Fb targeting each sequence were then analyzed.ALFA The expression plasmid was transfected into HEK293T cells, and the labeling results were observed using confocal microscopy 24 hours later.

[0135] To verify Bi-miniGFP-Fb ALFA The non-repetitive sequence imaging capability will target sgRNA, dCas9-24×ALFA, and Bi-miniGFP-Fb at the MUC4.1 site. ALFA The expression plasmid was transfected into HEK293T cells, and the labeling results were observed using confocal microscopy 24 hours later.

[0136] Table 8. sgRNA information targeting non-tandem repeat sequences

[0137] ;

[0138] Experimental results:

[0139] Figure 9A shows the microscopic results of labeling non-tandem repeat sequences on different chromosomes using the BRIGHT system. Two bright near-infrared fluorescent signal spots are visible in the cell nucleus, indicating that the new method can label non-tandem repeat sequences.

[0140] Figure 9B shows the use of Bi-miniGFP-Fb ALFA Microscopic results of the MUC4.1 site marker show two bright green fluorescent signal spots within the cell nucleus, indicating Bi-miniGFP-Fb. ALFA It can be used to label non-tandem repeating sequences.

[0141] Example 5: Validating the performance of the BRIGHT live-cell DNA non-repetitive sequence imaging system

[0142] ①Imaging specificity:

[0143] To verify the imaging specificity of non-repetitive sequences, co-localization imaging experiments were conducted using the BRIGHT system and Oligo-FISH. The BRIGHT system targeted the MUC4.1 non-tandem repeat sequence, while Oligo-FISH targeted MUC4Rep (oligonucleotide fluorescent probe: / Cy3 / CTTCCTGTCACCGAC).

[0144] The experimental steps are as follows:

[0145] BRIGHT live-cell DNA imaging:

[0146] a. Place a 20mm × 20mm coverslip at the bottom of a six-well plate and treat with poly-D-lysine to promote cell adhesion and growth. Seed HEK293T cells on the coverslip. When the confluence reaches 70%, transfect the cells according to the transfection system in Table 9 using jetPRIME transfection reagent (Polyplus) to transfect sgRNA targeting MUC4.1, dCas9-24×ALFA, and Bi-NIR-Fb. ALFA The expression plasmid was transfected into the cells described above.

[0147] Oligo-FISH:

[0148] b. 16 h after transfection, wash once with 1 mL of 1×PBS to remove dead cells.

[0149] c. Fix with 4% paraformaldehyde for 15 min (room temperature).

[0150] d. Wash three times with PBS for 5 minutes (at room temperature).

[0151] e. Use 2×SSC containing 0.7% Triton X-100 for 30 min (room temperature).

[0152] f. Wash cells twice with 2×SSC (at room temperature).

[0153] g. Treat with 100ug / mL RNase A at 37°C for 1h (37°C).

[0154] h. Wash once with 2×SSC for 5 minutes (room temperature).

[0155] i. Equilibrate in PBS for 5 min (at room temperature).

[0156] j. Dehydration: 70%, 85%, 100% for 2 minutes each, then air dry (room temperature).

[0157] k. Adjust the temperature of the StatSpin® ThermoBrite in situ hybridization instrument to 80°C, place the slide on the hybridization instrument, add 100 µL of 2×SSC containing 70% formamide to the slide, invert the cell coverslip onto the slide, and maintain at 80°C for 10 min to denature the sample.

[0158] l: Shake the slide vertically to remove the coverslip.

[0159] m: Incubate coverslips for 5 min each with 70%, 85%, and 100% ice-cold ethanol pre-cooled to -20℃ (on ice).

[0160] n: After removing ethanol and air-drying the sample, add 10µL of oligonucleotide fluorescent probe hybridization solution to the coverslip, invert it onto the slide, and seal it with rubber.

[0161] o: Set the hybridization instrument temperature to 37°C and place the slide on the hybridization instrument for overnight incubation.

[0162] After 16 hours, remove the coverslip and wash three times with 2×SSC solution in the dark (at room temperature).

[0163] q: Confocal observation was performed after DAPI staining.

[0164] Table 9 Transfection System

[0165] ;

[0166] Experimental results:

[0167] Figure 10 shows the colocalization experiment results. The BRIGHT imaging results are given red pseudocolor and the Oligo-FISH imaging results are given green pseudocolor. The colocalization signal of the two is yellow, indicating that the BRIGHT system can specifically label the MUC4.1 non-repetitive sequence.

[0168] ②Optimal conditions for visualizing non-repetitive sequences.

[0169] The key components of the BRIGHT imaging system are dCas9-n×ALFA and nanobodies with cascade effects, which are alternately linked to achieve exponential amplification of the fluorescence signal. To determine the optimal conditions for visualizing non-repetitive sequences, dCas9-n×ALFA (n=1, 4, 8, 16, 24) (500 ng) and Bi-NIR-Fb were used respectively. ALFA Bivalent nanobody (500 ng) and Tri-NIR-Fb ALFA Trivalent nanobodies (500 ng) were used to label MUC4.1 genomic loci. Images were acquired under identical imaging conditions for all experimental groups. The fluorescence intensity of 20 signal points in each group was quantitatively analyzed using ImageJ software. The Oval Selection tool was used to randomly select 30 identical circular regions (10 × 10 pixels) in the background to calculate the average background fluorescence intensity (Noise), thus calculating the SNR value. Using the Straight Line tool in ImageJ, lines were drawn along the central axis of representative signal points, and the Plot Profile tool was used to plot the fluorescence intensity as a function of the spatial distance of the drawn lines.

[0170] Experimental results:

[0171] Figure 11A shows the use of dCas9-n×ALFA (n=1, 4, 8, 16, 24) and Bi-NIR-Fb. ALFA and Tri-NIR-Fb ALFA The results of MUC4.1 genomic locus marker analysis are shown in the image, along with curves illustrating the fluorescence intensity of representative signal points as a function of spatial distance from the lines, obtained through Plot Profile analysis. This was achieved using Bi-NIR-Fb... ALFA During imaging, dCas9-1×ALFA, dCas9-4×ALFA, and dCas9-8×ALFA failed to successfully label the MUC4.1 site, while dCas9-16×ALFA and dCas9-24×ALFA successfully labeled the MUC4.1 site. This was achieved using Tri-NIR-Fb. ALFA During imaging, dCas9-1×ALFA and dCas9-4×ALFA failed to successfully label the MUC4.1 site, while dCas9-8×ALFA, dCas9-16×ALFA and dCas9-24×ALFA successfully labeled the MUC4.1 site.

[0172] Figure 11B shows the signal spot diameters of each group of marked MUC4.1 sites. (Bi-NIR-Fb) ALFA / dCas9-16×ALFA、Bi-NIR-Fb ALFA / dCas9-24×ALFA、Tri-NIR-Fb ALFA / dCas9-8×ALFA、Tri-NIR-Fb ALFA / dCas9-16×ALFA and Tri-NIR-Fb ALFA The average diameters of the signal points at the / dCas9-24×ALFA-labeled MUC4.1 sites were 0.97±0.16μm, 1.16±0.29μm, 0.65±0.18μm, 0.90±0.27μm, and 1.08±0.24μm, respectively.

[0173] Figure 11C shows the SNR values ​​of the MUC4.1 marker sites in each group. (Bi-NIR-Fb) ALFA / dCas9-16×ALFA、Bi-NIR-Fb ALFA / dCas9-24×ALFA、Tri-NIR-Fb ALFA / dCas9-8×ALFA、Tri-NIR-Fb ALFA / dCas9-16×ALFA and Tri-NIR-Fb ALFAThe mean SNRs of / dCas9-24×ALFA-labeled MUC4.1 sites were 52.91±19.14, 57.19±25.93, 19.92±8.70, 29.56±11.54, and 37.36±11.21, respectively. (Bi-NIR-Fb) ALFA The signal-to-noise ratio of the group was significantly higher than that of Tri-NIR-Fb. ALFA Group (p<0.01), while Bi-NIR-Fb ALFA / dCas9-16×ALFA and Bi-NIR-Fb ALFA There was no statistically significant difference in signal-to-noise ratio between the / dCas9-24×ALFA group (p>0.05).

[0174] In summary, Bi-NIR-Fb ALFA / dCas9-16×ALFA group and Bi-NIR-Fb ALFA The / dCas9-24×ALFA group can achieve high signal-to-noise ratio imaging of non-repetitive sequences.

[0175] Example 6: The BRIGHT system can be used for imaging non-repetitive sequences in various cell lines.

[0176] Tumor cell lines of different tissue origins and disease types were selected, including the human cervical cancer cell line HeLa, the human hepatocellular carcinoma cell line HepG2, and the human osteosarcoma cell line U2OS. Following the transfection system in Table 10, Lipofectamine 3000 transfection reagent (Thermo FISHer) was used to transfect sgRNA, dCas9-16×ALFA, and Bi-NIR-Fb targeting MUC4.1. ALFA The expression plasmid was transfected into the above cells, and the labeling results were observed using a confocal microscope 24 hours later.

[0177] Table 10 Transfection System

[0178] ;

[0179] The experimental steps are as follows:

[0180] a. HeLa, HepG2, and U2OS cells were plated and cultured overnight. Transfection was performed after the cell density reached 70%.

[0181] b. 30 minutes before transfection, replace the culture medium with 2 mL of Opti-MEM serum-reduced medium (phenol red-free, ThermoFISHer).

[0182] c. Dilute Lipofectamine™ 3000 reagent: Add 125µL of Opti-MEM™ culture medium to a 1.5 mL EP tube, followed by 7.5µL of Lipofectamine™ 3000 reagent, vortex for 2-3 seconds and then briefly dissociate.

[0183] d. Dilution of DNA: Add 125 µL of Opti-MEM™ medium to another 1.5 mL EP tube. Add each plasmid to 125 µL of Opti-MEM™ medium according to the plasmid transfection amounts in Table 10. Add the corresponding volume of P3000™ reagent at a ratio of total nucleic acid mass (µg): P3000™ reagent (µL) = 1:2. Vortex for 5 seconds and then briefly aliquot.

[0184] e. Mix 125µL of diluted Lipofectamine™ 3000 reagent with 125µL of diluted DNA, vortex for 2-3 seconds, and then briefly separate.

[0185] f. Incubate at room temperature for 15 min.

[0186] g. Slowly add 250µL of the transfection mixture to the culture dish, gently shake to mix, and incubate at 37°C for 4-6 hours.

[0187] h.4-6h later, replace the transfection medium with complete medium and return the plate to the incubator.

[0188] i. The results were observed using a confocal fluorescence microscope 24 hours after transfection.

[0189] Experimental results:

[0190] Figure 12 shows the microscopic results of MUC4.1 non-tandem repeat sequences labeled in HeLa, HepG2, and U2OS cells using the BRIGHT system. Three, two, and two signal points were observed in HeLa, HepG2, and U2OS cells, respectively, indicating that the new method can achieve imaging of non-repetitive sequences in different cell lines.

[0191] This invention is not limited to the structures described above and shown in the accompanying drawings, and various modifications and changes can be made without departing from its scope. The scope of this invention is limited only by the appended claims.

Claims

1. A live-cell DNA imaging system based on CRISPR and nanobodies, characterized in that, The invention includes a modified dCas9 protein expression vector, a modified sgRNA expression vector, and a fluorescent nanobody expression vector. The modified dCas9 protein expression vector contains dCas9 and n tandem ALFA polypeptide tags (n=1, 4, 8, 16, 24), and can bind to the corresponding nanobody. The modified sgRNA expression vector contains a specific guide sequence that pairs with the dCas9 protein and targets the detection target sequence. The modified dCas9 protein binds to the target nucleic acid under the guidance of the modified sgRNA. The antigen complementarity-determining region of the nanobody in the bivalent or trivalent fluorescent nanobody expression vector can recognize the tandem ALFA polypeptide tags in the modified dCas9 protein expression vector, and the fluorescent protein acts as an internal tag inserted between the nanobody cleavage sites. The fluorescent nanobody can alternately bind to the modified dCas9 protein to form a multi-layered cascade complex, thereby achieving a cascaded signal amplification reaction.

2. The live-cell DNA imaging system based on CRISPR and nanobodies according to claim 1, characterized in that, The modified dCas9 protein expression vector, the modified sgRNA expression vector, and the fluorescent nanobody expression vector were transfected into cell lines.

3. The live-cell DNA imaging system based on CRISPR and nanobodies according to claim 1, characterized in that, The modified dCas9 protein expression vector and fluorescent nanobody expression vector contain nuclear localization sequences when labeling nucleic acid sequences in the cell nucleus.

4. The live-cell DNA imaging system based on CRISPR and nanobodies according to claim 1, characterized in that, The fluorescent nanobody expression vector is driven by the CMV promoter, the EF-1α core promoter, the hPGK promoter, and the miniCMV promoter.

5. A live-cell DNA imaging system based on CRISPR and nanobodies according to claim 1, characterized in that, Fluorescent proteins inserted into fluorescent nanobody expression vectors are excited across the entire spectrum from ultraviolet to near-infrared to display imaging effects of different colors of fluorescence.

6. A live-cell DNA imaging method based on CRISPR and nanobodies for non-diagnostic purposes, characterized in that, include: Construct a live-cell DNA imaging system based on CRISPR and nanobodies as described in any one of claims 1-5; The components of the constructed CRISPR- and nanobody-based live-cell nucleic acid imaging system were transfected into the cells to be tested. This method utilizes live-cell imaging instruments to observe fluorescent signal spots formed by a CRISPR- and nanobody-based live-cell nucleic acid imaging system. It is used for imaging labeling of DNA in live cells, where the DNA includes repetitive and non-repetitive sequences. The method can achieve imaging of non-repetitive DNA sequences using the following combinations: a combination of a bivalent fluorescent nanobody expression vector and a dCas9 expression vector carrying 16 or 24 tandem ALFA peptide tags; or a combination of a trivalent fluorescent nanobody expression vector and a dCas9 expression vector carrying 8, 16, or 24 tandem ALFA peptide tags.

7. The use of the live-cell DNA imaging system based on CRISPR and nanobodies according to any one of claims 1-5 for the preparation of a kit.

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