A protein tagging system and its application

Abstract

This invention belongs to the field of protein tagging technology and discloses a protein tagging system and its applications. The protein tagging system of this invention includes an ST antigenic epitope and an AntiST antibody. The amino acid sequence of the ST antigenic epitope is shown in SEQ ID NO: 1; the AntiST antibody includes a light chain and a heavy chain; the amino acid sequence of the light chain is shown in SEQ ID NO: 3, and the amino acid sequence of the heavy chain is shown in SEQ ID NO: 5. The novel ST protein tagging system of this invention has high specificity, expanding the types of protein tagging systems; it can meet the needs of protein fluorescence imaging, realizing protein visualization; and it is also compatible with various base editing technologies, enabling various types of base mutations.

Description

Technical Field

[0001] This invention relates to the field of protein tagging technology, and more specifically to a protein tagging system and its application. Background Technology

[0002] Protein-tagging systems are tools for studying protein-protein interactions, enabling the specific labeling and capture of target proteins within cells. A typical protein recruitment system includes a short, identifiable tag, an antigenic epitope, approximately 10 to 30 amino acids in size. The system also includes a corresponding antibody that specifically binds to these epitopes, forming a stable antigen-antibody complex. By binding the epitope to the target protein and then fusing its antibody with another protein, the two proteins are brought close together spatially, allowing the target protein to be labeled for efficient enrichment and visualization. The main applications of protein-tagging systems are: 1) Capture and enrichment: By adding antibodies with capture tags (such as His and Flag tags), target proteins containing antigenic epitopes can be captured in cells or in vitro. This capture can be achieved through methods such as affinity chromatography and immunoprecipitation. 2) Protein fluorescence imaging: Using fluorescently labeled antibodies (such as fluorescent proteins), the localization and dynamic changes of target proteins can be observed and imaged. Using fluorescence microscopy, researchers can monitor the behavior and interactions of proteins within cells in real time. Protein tagging systems allow for real-time tracking of the dynamic changes of specific proteins within cells, enabling the study of their roles in processes such as cell cycle, signal transduction, and cell migration. 3) Gene Editing: In gene editing technologies such as CRISPR / Cas, protein tagging systems can be fused with Cas proteins and other related proteins for expression, achieving modularity in gene editing technology. Protein tagging systems contribute to the controllability of gene regulation. They provide powerful tools for studying and manipulating biomolecules, playing a crucial role, particularly in protein fluorescence imaging and gene editing. Protein tagging systems allow scientists to precisely manipulate and observe protein function and interactions at the molecular level, and are widely used to study protein-protein interactions, intracellular localization, and the tracking of dynamic processes.

[0003] Currently, protein recruitment systems include: (1) SunTag system: SunTag is a common protein tagging system containing an antigenic epitope GCN4 of 19 amino acids and an antibody scFv (single-chain variable fragment antibody) of 270 amino acids. GCN4 was originally derived from Saccharomyces cerevisiae and is an important domain of the yeast transcription activator General Control Non-derepressible 4. GCN4 contains a specific amino acid sequence that can form a specific three-dimensional structure and can be used as a recognized epitope. scFv is an antibody fragment artificially constructed through genetic engineering technology. The heavy chain variable region (VH) and light chain variable region (VL) of scFv are linked by a flexible linker peptide. VH and VL contain the key regions for antibody recognition of antigens, namely complementarity-determining regions (CDRs). scFv retains the antibody's specific recognition ability for antigens and can bind to GCN4 with high affinity. Its small molecular weight and single-chain structure give it several unique advantages, such as easier intracellular expression and tissue penetration, and greater flexibility in fusing with other molecules. Furthermore, GCN4 can be tandemly repeated to form multiple binding sites, facilitating the simultaneous binding of multiple antibodies and enhancing recruitment. In the SunTag system, scFv acts as a crucial bridge, serving as an antibody fragment that recognizes GCN4. It accurately guides effector molecules (such as fluorescent proteins and therapeutic proteins) to target proteins or regions containing GCN4 epitopes, enabling labeling, detection, and regulation of targets. For example, in protein fluorescence imaging, fluorescent proteins are fused with scFv; scFv, through binding to GCN4, carries the fluorescent protein to the target protein, making the target protein visible and facilitating the study of its intracellular distribution, dynamic changes, and other biological characteristics. Simultaneously, scFv can also be fused with other biologically active molecules to regulate target protein function or participate in gene editing. Binding scFv to transcription factors such as VP64 and GCN4 to dCas9 can achieve gene overexpression. In recent years, the SunTag system has also been gradually used in cytosine base editing technology (a category of base editing technology). By fusing GCN4 with nCas9 (D10A) protein and fusing scFv with deaminase APOBEC1, base editing can be achieved, that is, cytosine C is modified into thymine T. (2) MoonTag system: The MoonTag system includes the antigenic epitope gp41 and the antibody Nbgp41. Similar to SunTag, MoonTag uses specific antibodies to recognize and bind to its tag, forming a stable antigen-antibody complex. gp41 is derived from human immunodeficiency virus (HIV).In the HIV viral envelope structure, gp41 is an important subunit of the envelope protein complex. In the MoonTag system, the gp41 epitope is generally a specific amino acid sequence of 15 amino acids, which functions to provide a site for specific binding to the antibody Nbgp41. Driven by the needs of HIV-related research and protein labeling and tracking technologies, researchers have developed a 123-amino acid-sized Nbgp41 nanobody capable of specifically recognizing the gp41 epitope. It was obtained through screening and cloning of an antibody library generated after immunization of camels, followed by a series of optimizations and identifications. The structure of Nbgp41 is relatively simple, containing only the variable region (VHH) of the heavy chain antibody. The VHH structure has a unique three-dimensional conformation, containing three complementarity-determining regions (CDRs): CDR1, CDR2, and CDR3. These CDR regions together constitute the antigen-binding site, determining the specificity and affinity of Nbgp41 for the gp41 epitope. Compared with traditional antibodies, nanobodies have some unique advantages, such as small molecular weight, high stability, and strong affinity. When studying certain biological processes in cells, gp41 epitopes can be fused with target proteins for expression, and Nbgp41 can be coupled with fluorescent proteins or other effector molecules. Nbgp41 can recruit these effector molecules to target proteins containing gp41 epitopes by binding to gp41 epitopes, thereby achieving the purpose of visualizing or regulating the function of target proteins. However, there are currently no literature reports on the use of the MonnTag system for gene editing technology. (3) ALFA system: The ALFA system consists of ALFA epitopes and corresponding antibodies NbALFA. The ALFA epitope is a small and strong tag that can specifically bind to NbALFA. This high affinity and specificity make the ALFA system very effective in labeling and capturing target proteins. ALFA is an artificially designed short peptide tag with a unique amino acid sequence that can be recognized and bound by specific nanobodies NbALFA. The labeling and recruitment of target proteins are achieved through the interaction between the ALFA tag and NbALFA. Unlike other protein tagging systems, NbALFA can fuse effector molecules with proteins, thereby recruiting the effector molecules to target proteins tagged with ALFA to perform their functions. Currently, ALFA systems are mainly used for protein imaging tracking and protein capture, and there are no reports in the literature regarding their application in gene editing technology.

[0004] However, the types of protein tagging systems currently available are limited. Each protein tagging system has its own limitations in application, and increasing the number of protein tagging systems would help expand their application scenarios. There are only reports in the literature regarding the use of the SunTag tagging system for gene editing, which may be because other protein tagging systems are not suitable for gene editing technologies. Currently reported applications of the SunTag system mainly involve cytosine base editing, while other base editing technologies include adenine base editing and glycosylation enzyme base editing. This means that the application of protein tagging systems in base editing technology is quite limited. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a protein tagging system and its application.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a protein tagging system comprising an ST antigen epitope and an AntiST antibody, wherein the amino acid sequence of the ST antigen epitope is shown in SEQ ID NO: 1; the AntiST antibody comprises a light chain and a heavy chain; the amino acid sequence of the light chain is shown in SEQ ID NO: 3, and the amino acid sequence of the heavy chain is shown in SEQ ID NO: 5.

[0008] This invention develops a novel protein tagging system, named Protein Tag System ST. The key antigenic epitope of the VP1 coat protein of human enterovirus 71 is named ST, serving as the key region for antibody recognition. A specific antibody targeting the shortest VP1 antigenic epitope is named AntiST, a bivalent neutralizing monoclonal antibody capable of simultaneously binding to two adjacent antigenic epitopes. This novel protein tagging system ST exhibits high specificity, expanding the range of protein tagging systems; it meets the requirements of protein fluorescence imaging, enabling protein visualization; and it is compatible with various base editing technologies, allowing for multiple types of base mutations.

[0009] In a preferred embodiment of the protein tagging system of the present invention, the light chain and heavy chain of the AntiST antibody are linked by a linker peptide; the amino acid sequence of the linker peptide is shown in SEQ ID NO: 4.

[0010] In a preferred embodiment of the protein tagging system of the present invention, the nucleotide sequence of the ST antigen epitope is shown in SEQ ID NO: 2; and the nucleotide sequence of the AntiST antibody is shown in SEQ ID NO: 6.

[0011] In a second aspect, the present invention provides a protein fluorescence imaging system, including the protein tagging system described above; the ST antigen epitope is fused with fluorescent protein and / or histone for expression; and the AntiST antibody is fused with fluorescent protein for expression.

[0012] The ST system of this invention, when fused with different fluorescent proteins, can impart different fluorescence to the target protein. The ST system exhibits extremely high specificity in protein fluorescence imaging. Furthermore, it can visualize proteins, such as H2B, thereby enabling visualization of the cell nucleus and chromosomes.

[0013] In a preferred embodiment of the protein fluorescence imaging system of the present invention, the fluorescent proteins include red fluorescent protein mcherry and green fluorescent protein sfGFP; the histones include H2B.

[0014] Thirdly, the present invention provides a gene editing system, including the protein tagging system described above; the ST antigen epitope is fused with nCas9(D10A) protein for expression; and the AntiST antibody is fused with deaminase for expression.

[0015] This invention integrates the ST system with base editing technology to achieve multiple base mutation methods, which is impossible with previous protein tagging systems. The ST system has strong compatibility and can be compatible with different base editing technologies, including but not limited to the base editing technologies mentioned in the embodiments of this invention.

[0016] In a preferred embodiment of the gene editing system of the present invention, the deaminase includes at least one of APOBEC1, TadA8eWQ, R33A, pmCDA1, and TadA9.

[0017] Fourthly, the present invention applies the protein tagging system and the protein fluorescence imaging system to protein fluorescence imaging and protein visualization.

[0018] Fifthly, the present invention relates to the application of the protein tagging system and the gene editing system in gene editing.

[0019] As a preferred embodiment of the application described in this invention, the gene editing includes at least one of editing the base C of the target site sequence to the base T, editing the base C of the target site sequence to the base G, and editing the base A of the target site sequence to the base G.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] (1) This invention provides a novel protein tagging system ST, which further expands the types of protein tagging systems and broadens the application scenarios of protein tagging systems.

[0022] (2) The protein tagging system ST of this invention can achieve protein visualization with high specificity. The ST system has a powerful and highly specific recruitment capability, and can recruit effector molecules (such as fluorescent proteins and various deaminases) to the target protein site through AntiST as a bridge, thereby realizing protein visualization or gene editing. It can not only achieve visualization of the cell nucleus, but also efficiently achieve visualization of chromosomes, which fully demonstrates the high efficiency and high specificity of the ST system's recruitment capability.

[0023] (3) The protein tagging system of the present invention has strong ST compatibility. It is compatible not only with fluorescent proteins, but also with a variety of base editing technologies, including cytosine base editing technology, adenine base editing technology and glycosylation enzyme base editing technology, so as to achieve a variety of base mutation types. Attached Figure Description

[0024] Figure 1 The images show protein fluorescence imaging of the ST protein tagging system. In the figure, A shows the plasmid structure used when the ST tag is located at the N-terminus of the fusion protein; B shows the protein fluorescence imaging when the ST tag is located at the N-terminus of the fusion protein. Red and green fluorescence are simultaneously observed in the cell nucleus and chromosomes. C shows the plasmid structure used when the ST tag is located at the C-terminus of the fusion protein; D shows the protein fluorescence imaging when the ST tag is located at the C-terminus of the fusion protein. Red and green fluorescence are simultaneously observed in the cell nucleus and chromosomes.

[0025] Figure 2 This is a schematic diagram of the structure of BE3 and ST-BE3.

[0026] Figure 3 The figure compares the editing efficiency of the BE3 and ST-BE3 systems at four target sites. The numbers in the small squares represent the percentage efficiency of the current C base mutation to T.

[0027] Figure 4 Schematic diagrams for ABE8eWQ and ST-ABE8eWQ.

[0028] Figure 5 A comparison of editing efficiency between .ABE8eWQ and ST-ABE8eWQ; the figure shows the sequences of the four target sites, and the numbers in the small squares represent the percentage efficiency of the current A base mutation to G.

[0029] Figure 6 Schematic diagrams of miniCGBE1 and ST-miniCGBE1.

[0030] Figure 7 A comparison of the editing efficiency of miniCGBE1 and ST-miniCGBE1 is shown in the figure; the sequence of the four target sites is displayed, and the number in the small square represents the percentage efficiency of the current C base mutation to G. Detailed Implementation

[0031] This invention develops a novel protein tagging system, named Protein Tag System ST, with its antigenic epitope and antibody named ST and AntiST, respectively. The key antigenic epitope of the capsid protein VP1 of human enterovirus 71 is named ST, serving as the key region for antibody recognition. This epitope typically forms a β-sheet-rich region, enabling stable binding to the antibody. This epitope may involve multiple interaction mechanisms, including hydrogen bonding, electrostatic interactions, and hydrophobic interactions. The specific antibody for the shortest VP1 epitope was selected and named AntiST. AntiST belongs to the IgG class of antibodies and possesses a complete antibody structure, including heavy and light chains. The variable regions of its heavy and light chains play a crucial role in recognizing the epitope, with the binding of the heavy chain CDR3 region to the epitope being particularly important. AntiST is a bivalent neutralizing monoclonal antibody capable of simultaneously binding to two adjacent epitopes, which is highly beneficial for protein recruitment.

[0032] The sequence information is as follows:

[0033] The amino acid sequence of the ST antigenic epitope (SEQ ID NO: 1): GYPTFGEHKQEKDLEYG; The base sequence of the ST antigenic epitope (SEQ ID NO: 2):

[0034] GGGTACCCCACATTCGGAGAACACAAACAGGAAAAAGACCTGGAGT ACGGA;

[0035] The amino acid sequence of the AntiST antibody:

[0036] MDVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQS PKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEADDVGVYYCYQGSHVPY TFGGGTKLEI (SEQ ID NO: 3, light chain),

[0037] GGGGSGGGGSGGGGS (SEQ ID No. 4, linker peptide),

[0038] EVQLQQSGAELVKPGASVKLSCTASGFNIKDTYIHWVKQRPEQGLEWIG KIDPANGNTKYDPKFQDKATITADTSSNTAYLQLSSLTSEDTAVYYCANSNYW FDFDYWGQGTTLTVS (SEQ ID NO: 5, heavy chain);

[0039] Base sequence of the AntiST antibody (SEQ ID NO: 6):

[0040] ATGGATGTGCTGATGACCCAGACCCCCCTGTCGCTGCCCGTGAGCCTGGGCGACCAGGCCTCTATTAGCTGCAGAAGCAGCCAGAGCATCGTGCACAGCAATGGCAACACCTACCTGGAATGGTATCTGCAGAAGCCTGGCCAGTCTCCTAAACTGCTGATCTATAAAGTGTCTAATAGGTTTAGCGGCGTGCCCGACCGCTTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGAAGATCAGCCGGGTGGAGGCCGACGATGTGGGCGTGTACTACTGCTACCAGGGCAGCCACGTTCCCTACACCTTCGGCGGCGGCACCAAGCTGGAGATCGGCGGCGGCGGCTCCGGGGGCGGCGGCAGCGGCGGAGGCGGCAGCGAGGTGCAGCTCCAGCAGAGCGGAGCAGAGCTGGTGAAGCCCGGAGCCAGCGTGAAGCTGAGCTGCACCGCCAGCGGCTTCAACATCAAGGACACCTACATCCACTGGGTGAAGCAGCGGCCTGAGCAGGGCCTGGAGTGGATCGGCAAGATTGACCCAGCCAACGGCAACACTAAGTATGACCCTAAGTTTCAGGATAAGGCCACTATAACCGCTGACACATCTAGTAATACCGCATATCTGCAGCTTAGTAGCCTGACCTCTGAGGATACGGCCGTCTATTACTGTGCTAATAGTAACTACTGGTTTGACTTTGATTACTGGGGCCAGGGCACCACACTGACCGTGAGC.

[0041] To apply ST antigenic epitopes and AntiST antibodies to practical protein recruitment systems and in protein fluorescence imaging and base editing technologies, it is necessary to optimize the connection method and efficiency between the antibody and the fluorescent protein or gene editing tool, and improve the stability and specificity of the system. To better illustrate the purpose, technical solution, and advantages of this invention, specific embodiments will be provided below for further explanation. Those skilled in the art should understand that the specific embodiments described herein are merely illustrative and not intended to limit the scope of the invention.

[0042] Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0043] Example 1: Protein fluorescence imaging of the ST protein tagging system

[0044] 1. Constructing fluorescent plasmids

[0045] Key components of the plasmid: ampicillin resistance gene, ori replicon, CMV promoter, and protein expression sequence, such as... Figure 1 As shown in A and C, histone and red fluorescent protein mcherry are fused together, while the ST tag is located at the N-terminus or C-terminus of the fusion protein; AntiST is fused to the N-terminus of green fluorescent protein and incorporates a GB1 protein element; the GB1 protein element helps alleviate spontaneous antibody aggregation and precipitation. All three plasmids were synthesized by Genewiz. The structures of the main plasmid elements are as follows:

[0046] The base sequence of ST-H2B-mcherry (SEQ ID NO: 7, the uppercase base sequence is the ST tag, ST is located at the N-terminus of the fusion protein):

[0047]

[0048] The base sequence of H2B-mcherry-ST (SEQ ID NO: 8, the uppercase base sequence is the ST tag, ST is located at the C-terminus of the fusion protein):

[0049]

[0050] The base sequence of AntiST-sfGFP (SEQ ID NO: 9, uppercase base sequence is AntiST, AntiST is located at the N-terminus of the fusion protein):

[0051]

[0052] 2. Fluorescent plasmid transfection

[0053] Human cervical cancer HeLa cell lines were co-transfected with fluorescent plasmids containing ST and AntiST, respectively. The specific steps are as follows:

[0054] (1) Cell culture: Cells were cultured in DMEM complete medium containing 10% serum at 37°C and 5% CO2. When the cell confluence reached 90%, the cells were digested with 0.25% trypsin and then the digestion was stopped with DMEM complete medium. The cells were then seeded into 12-well plates and cultured for another 24 hours.

[0055] (2) Plasmid transfection: After 24 hours, once cell adhesion was confirmed to be good and cell confluence reached 80%, transfection could be performed. Transfect each well with 0.5 μg of ST plasmid and 0.5 μg of AntiST plasmid. Transfection was performed using Yeasen's Polyethylenimine Linear (PEI) MW40000 transfection reagent according to the manufacturer's instructions. Transfected cells were then cultured at 37°C in a 5% CO2 incubator.

[0056] 3. Protein fluorescence imaging

[0057] Forty-eight hours post-transfection, cell nuclei and chromosomes were imaged using a laser confocal microscope. To further clarify the presence of genomic DNA, cell samples were stained with Dapi blue fluorescent dye.

[0058] The green fluorescent protein sfGFP was fused with the antibody AntiST for expression, and the ST tag was fused with the red fluorescent protein mcherry and histone H2B for expression. H2B specifically localizes to the cell nucleus, and because mcherry and H2B are fused, the cell nucleus appears red. Simultaneously, the ST tag is fused with both H2B and mcherry, thus recruiting the AntiST-sfGFP fusion protein into the cell nucleus. Ultimately, this results in the cell nucleus appearing both red and green. Figure 1 As shown in B and D, ST, located at either the N-terminus or C-terminus of the H2B-mcherry fusion protein, can specifically recruit AntiST-sfGFP to the cell nucleus. When cells begin mitosis and form distinct chromosomes, both red and green fluorescence are specifically present on the chromosomes, further demonstrating that the ST tag can highly specifically recruit the antibody AntiST.

[0059] Example 2: Cytosine base editing technology based on ST tag system

[0060] 1. Genomic target sites

[0061] The primers used were synthesized by Suzhou Genewiz Biotechnology Co., Ltd.; the PCR reagents were from Beijing TransGen Biotech Co., Ltd. PCR SuperMix, catalog number AS111-02. To illustrate the base editing potential of the novel protein tagging system ST of this invention, the following examples demonstrate base editing at human genomic loci. Those skilled in the art can design base editing schemes for other gene loci based on the following examples.

[0062] Table 1 shows the target site sequences used in this embodiment and their editing results. Target editing results: (1) Edit the base C of the target site sequence to the base T; (2) Edit the base C of the target site sequence to the base G; (3) Edit the base A of the target site sequence to the base G.

[0063] Table 1 Target sites and their editing methods

[0064]

[0065] 2. Construct sgRNA plasmids targeting the target site

[0066] The main components of the sgRNA plasmids were: ampicillin resistance gene, ori replicon, U6 promoter, spacer sequence, and scaffold backbone sequence. The template plasmid was purchased from the Miaoling plasmid platform (catalog number P11694). Spacer sequences from the HEK3, FANCF, ZAP70, and VEGFA sites were used to construct sgRNA plasmids. DNA fragments were obtained by PCR amplification. Primer 1 contained the 20-nt sequence at the 3' end of the U6 promoter, the 20-nt sequence of the spacer, and the 20-nt sequence at the 5' end of the scaffold. Primer 2 was the reverse complementary sequence of the 20-nt sequence at the 3' end of the U6 promoter. Primers were synthesized by Genewiz and then cloned into the plasmid vector using the Gibson assembly method. The constructed plasmids were named HEK3-sgRNA, FANCF-sgRNA, ZAP70-sgRNA, and VEGFA-sgRNA. After construction, the correct sgRNA plasmid sequence was confirmed to be free of mutations by routine sequencing comparison. Single colonies with completely correct sequences were selected for amplification and plasmid extraction.

[0067] The target site Spacer sequence is shown in Table 1;

[0068] Scaffold sequence (SEQ ID NO: 20):

[0069] GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCC;

[0070] Primer 1 (SEQ ID NO: 21):

[0071] GTGGAAAGGACGAAACACCG (Spacer 20nt sequence)GTTTTAGAGCTAGAAATAGC;

[0072] Primer 2 (SEQ ID NO: 22): CGGTGTTTCGTCCTTTCCAC.

[0073] 3. Positive control plasmid

[0074] The BE3 plasmid was purchased from the Miaoling plasmid platform, catalog number P1236. The BE3 plasmid contains an nCas9 (D10A) sequence with DNA single-strand cutting activity, an APOBEC1 editing enzyme sequence that enables the editing of base C to T, and a UGI sequence to improve editing efficiency.

[0075] 4. Construction of ST-BE3 plasmid.

[0076] The APOBEC1 and UGI sequences were derived from plasmid pST1374-scFv-APOBEC-UGI-GB1, purchased from Addgene (catalog number 113029). The nCas9(D10A) and nuclear localization signal peptide sequences were derived from plasmid pST1374-GCN4-D10A, purchased from Addgene (catalog number 113022). The antigenic epitope ST was fused to the N-terminus of nCas(D10A) and named D10A-ST; the antibody AntiST was fused with APOBEC1 and named AntiST-BE3. The two plasmids were combined and named ST-BE3. Figure 2 As shown. After construction, the plasmid sequence was confirmed to be correct and without mutations by routine sequencing comparison. Single colonies with completely correct sequences were selected for amplification and plasmid extraction.

[0077] The sequence information for the ST-BE3 system is as follows:

[0078] The base sequence of D10A-ST (SEQ ID NO: 23, the uppercase base sequence is the ST sequence, "GCT(D10A)" indicates that the 10th amino acid of nCas9(D10A) has mutated from D to A):

[0079]

[0080] AntiST-BE3 base sequence (SEQ ID NO: 24, AntiST antibody is represented by uppercase base sequence):

[0081]

[0082] 4. Compare the editing characteristics of BE3 and ST-BE3

[0083] Human embryonic kidney 293T cell lines were co-transfected with sgRNA plasmids HEK3-sgRNA, FANCF-sgRNA, ZAP70-sgRNA, and VEGFA-sgRNA along with the ST-BE3 double plasmid. Control groups were established using either the sgRNA plasmid or the BE3 plasmid. Editing characterization was accurately assessed using sequencing.

[0084] The specific steps are as follows:

[0085] (1) Cell culture: Human embryonic kidney cell line 293T was cultured in DMEM complete medium containing 10% serum at 37°C and 5% CO2 in an incubator. When the cell confluence reached 90%, it was digested with 0.25% trypsin and then digested with DMEM complete medium to stop the digestion. The cells were then seeded into 12-well plates and cultured for another 24 hours.

[0086] (2) Plasmid transfection: After 24 hours, once cell adhesion was confirmed to be good and cell confluence reached 80%, transfection could be performed. Each well was transfected with 0.5 μg of sgRNA plasmid, 0.5 μg of D10A-ST plasmid, and 0.5 μg of AntiST-BE3 plasmid. Transfection was performed using Yeasen's Polyethylenimine Linear (PEI) MW40000 transfection reagent according to the manufacturer's instructions, with 1 μg of BE3 plasmid used as a control group. Transfected cells were then cultured at 37°C in a 5% CO2 incubator.

[0087] (3) Extraction of genomic DNA: 48 hours after transfection, cells were routinely digested with 0.25% trypsin and digestion was terminated with DMEM complete medium. Cells were collected into centrifuge tubes, centrifuged at 300g for 5 minutes, the medium was discarded, washed once with PBS, centrifuged again at 300g for 5 minutes, the PBS was discarded, and cell residue was obtained. Genomic DNA was extracted from the cells using a cell genomic DNA extraction kit (TransGen Biotech Ltd., catalog number: EE101-01), and the DNA concentration was measured.

[0088] (4) Genomic PCR: Based on the genomic sequences of HEK3, FANCF, ZAP70, and VEGFA, upstream and downstream primers were designed, and the specific sequences are shown in Table 2. After amplifying the target DNA product, the product was sent to Guangzhou Sangon Biotech Co., Ltd. for sequencing.

[0089] Table 2 Primer sequences

[0090]

[0091] The Sanger sequencing results were analyzed to determine the editing efficiency of ST-BE3 at four target sites. The results are as follows: Figure 3 As shown, ST-BE3 can significantly edit bases at these four target sites, achieving C-to-T mutations, and its efficiency is significantly higher than that of the original base editor BE3. In base editing technology, the ST tag is fused to nCas9(D10A), and AntiST is fused to a deaminase, thereby achieving modularity of base editing technology, which is beneficial for gene regulation.

[0092] Example 3: Adenine base editing technology based on ST tag system

[0093] Analysis of the editing results in Example 2 revealed that the ST-BE3 system exhibited powerful and superior editing performance. To further expand the types of base editing technologies compatible with ST, the base editor ABE8eWQ was introduced, and the ST-BE8eWQ system was constructed.

[0094] 1. Constructing sgRNA plasmids targeting specific sites

[0095] The sgRNA plasmid construction protocol is the same as in Example 2. The constructed plasmids are named: HEK3-sgRNA, HEK2-sgRNA, OCT4-sgRNA, and ZAP70-sgRNA.

[0096] 2. Positive control plasmid

[0097] The ABE8eWQ plasmid was purchased from Addgene, catalog number 161815. The ABE8eWQ plasmid contains the nCas9(D10A) sequence with DNA single-strand cutting activity and the TadA8eWQ editing enzyme sequence that enables the editing of base A to G.

[0098] 3. Construction of ST-ABE8eWQ plasmid

[0099] The construction scheme is the same as in Example 2. The TadA8eWQ sequence is derived from the ABE8eWQ plasmid. AntiST was fused with ABE8eWQ and expressed, named AntiST-ABE8eWQ, thus establishing the ST-ABE8eWQ system. The results are shown in [Figure 1]. Figure 4 After construction, the plasmid sequence was confirmed to be correct and without mutations by routine sequencing comparison. Single colonies with completely correct sequences were selected for amplification and plasmid extraction.

[0100] The base sequence of AntiST-ABE8eWQ (SEQ ID NO: 33, uppercase base sequence is AntiST):

[0101]

[0102] 4. Compare the editing characteristics of ABE8eWQ and ST-ABE8eWQ

[0103] Human embryonic kidney 293T cell lines were co-transfected with sgRNA plasmids HEK3-sgRNA, HEK2-sgRNA, OCT4-sgRNA, and ZAP70-sgRNA along with the ST-ABE8eWQ double plasmid. A control group was prepared by transfecting cells with either the sgRNA plasmid or the ABE8eWQ plasmid. Sequencing was performed using the same protocol to accurately assess the editing efficiency.

[0104] The results are as follows Figure 5 As shown, ST-ABE8eWQ can efficiently perform base editing at these four target sites, achieving the mutation of A into G.

[0105] Example 4: Glycosylation Enzyme Base Editing Technology Based on ST Tag System

[0106] To further verify whether the ST tagging system can be compatible with more base editing technologies, the base editor miniCGBE1 was introduced, and the ST-miniCGBE1 system was built.

[0107] 1. Constructing sgRNA plasmids targeting specific sites

[0108] The sgRNA plasmid construction protocol is the same as in Example 2. The constructed plasmids are named HEK3-sgRNA, FANCF-sgRNA, EMX1-sgRNA, and PPP1R12C-sgRNA.

[0109] 2. Positive control plasmid

[0110] The miniCGBE plasmid was purchased from Addgene, catalog number 140253. The miniCGBE1 plasmid contains the nCas9(D10A) sequence with DNA single-strand cutting activity and the R33A editing enzyme sequence that enables the editing of base C to G.

[0111] 3. Construction of ST-miniCGBE1 plasmid

[0112] The construction scheme is the same as in Example 2. The R33A sequence is derived from the miniCGBE1 plasmid. The antibody AntiST is fused with miniCGBE1 and expressed, named AntiST-miniCGBE1, thus establishing the ST-miniCGBE1 system. (See [link to documentation]). Figure 6 After construction, the plasmid sequence was confirmed to be correct and without mutations by routine sequencing comparison. Single colonies with completely correct sequences were selected for amplification and plasmid extraction.

[0113] The base sequence of AntiST-miniCGBE1 (SEQ ID NO: 34, uppercase base sequence is AntiST):

[0114]

[0115] 4. Compare the editing characteristics of miniCGBE1 and ST-miniCGBE1

[0116] Human embryonic kidney 293T cell lines were co-transfected with sgRNA plasmids HEK3-sgRNA, FANCF-sgRNA, EMX1-sgRNA, and PPP1R12C-sgRNA along with the ST-miniCGBE1 double plasmid. Control groups were prepared by transfecting cells with either sgRNA or miniCGBE1 plasmids. Sequencing was performed using the same protocol to accurately assess the editing efficiency.

[0117] The results are as follows Figure 7 As shown, ST-miniCGBE1 can achieve base editing at these four target sites, enabling C mutations to G.

[0118] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A protein tagging system, characterized in that, The fusion protein includes a fusion protein containing an ST antigen epitope and a fusion protein containing an AntiST antibody, wherein the nucleotide sequence of the fusion protein containing the ST antigen epitope is shown in SEQ ID NO: 7 or 8; and the nucleotide sequence of the fusion protein containing the AntiST antibody is shown in SEQ ID NO:

9.

2. A protein fluorescence imaging system, characterized in that, Includes the protein tagging system of claim 1.

3. The protein fluorescence imaging system according to claim 2, characterized in that, The fluorescent proteins include red fluorescent protein mcherry and green fluorescent protein sfGFP; the histones include H2B.

4. A gene editing system, characterized in that, The invention includes a fusion protein containing an ST antigen epitope and a fusion protein containing an AntiST antibody; the amino acid sequence of the ST antigen epitope is shown in SEQ ID NO: 1; the nucleotide sequence of the AntiST antibody is shown in SEQ ID NO:

6. The fusion protein containing the ST antigen epitope consists of the ST antigen epitope, nCas9D10A, and NLS, from segment N to segment C, respectively. The fusion protein containing the AntiST antibody can be any of the following from segment N to segment C: i. AntiST antibody, APOBEC1, UGI, GB1, NLS; ii. AntiST antibody, TadA8eWQ, GB1, NLS; iii. AntiST antibody, R33A, GB1, NLS.

5. The application of the protein tagging system of claim 1, or the protein fluorescence imaging system of claim 2 or 3, in protein fluorescence imaging and protein visualization.

6. The application of the protein tagging system of claim 1 and the gene editing system of claim 4 in gene editing for purposes other than disease diagnosis or treatment.

7. The application according to claim 6, characterized in that, The gene editing includes at least one of editing the base C of the target site sequence to the base T, editing the base C of the target site sequence to the base G, and editing the base A of the target site sequence to the base G.

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