Construct for real-time tracing of spatial genome conformation and transcription products in living cells and use thereof

Abstract

The present invention relates to a construct for the real-time tracing of spatial genome conformation and transcription products in living cells and the use thereof. Specifically, provided is a construct, which is a multifunctional DNA and RNA imaging tool (abbreviated to DRIT). The construct is used in the design / preparation method of transgenic animals, and is used for real-time in-situ tracing of spatial genome conformation and labeling and tracing of nascent transcription products in living cells.

Description

Constructs for real-time tracking of genome spatial conformation and transcription products in living cells and their uses Technical Field

[0001] The present disclosure relates to the fields of biology, medicine, clinical medicine, and imaging. Specifically, the present disclosure provides a novel imaging technique, DRIT, for tracing chromatin loop structures and nascent transcripts in living cells. Background Art

[0002] The molecular mechanisms underlying cell fate determination during individual development are challenging scientific questions in medicine and life sciences. The billions of cells that make up the human body differentiate from a single fertilized egg. While each cell contains identical genetic information—its genomic DNA—they comprise hundreds of distinct cell types, forming organs that perform diverse physiological functions. The challenging question is: how does a single fertilized egg differentiate into these hundreds of cell types during individual development? And what are the molecular mechanisms underlying changes in cellular identity and function under pathological conditions and during the development of disease?

[0003] Research has shown that cell fate determination during individual development and disease progression is regulated by lineage-specific gene expression. The lineage-specific nature of gene expression is meticulously regulated at multiple levels: the three-dimensional (3D) conformation of genomic DNA at the chromatin level, epigenetic modifications of genomic DNA and histones, and regulation by transcription factors and cofactors. Genomic DNA, the carrier of genetic information, binds to histones and folds into a highly ordered three-dimensional structure within the cell nucleus. While the genomic DNA sequence is identical in different cell types, its 3D conformation varies, playing a crucial role in gene expression regulation. Therefore, understanding human genome function, gene expression regulation, cell fate determination, and disease mechanisms requires not only understanding the one-dimensional linear DNA sequence but, more importantly, uncovering the 3D conformation of genomic DNA and its dynamic changes. Deciphering the temporal (four-dimensional) conformation of genomic DNA is key to understanding genome function, gene expression regulation, and the causal relationship between genomic DNA and cell fate determination.

[0004] In the past decade, with the development of chromatin conformation capture (3C) and its derivative technologies, as well as various genomic DNA imaging technologies based on fluorescence in situ hybridization (FISH), people have depicted the high-level chromatin structures at different scales: chromosome territories, A / B compartments, topologically associating domains (TADs) and chromatin loops.

[0005] Although existing technologies can analyze the three-dimensional structure of chromatin / genomic DNA, most of them are based on snapshot approaches, ignoring higher-dimensional features such as time scale and chromatin structural dynamics. During development, the fate of different cell lineages is closely related to the temporal and spatial changes of chromatin structure and corresponding transcription factors. Therefore, studying the high-dimensional structure of chromatin in a higher dimension is crucial for us to understand its causal relationship with gene expression regulation and developmental differentiation processes. However, the limitations of existing genomic DNA imaging technology are: (1) It is necessary to modify genomic DNA - add imaging-related sequences, construct modified cell lines, and transfect cells with imaging elements for imaging. Such methods are very unfriendly to primary cultured tissue cells. Primary cell transfection is difficult, and it is impossible to construct a stably transfected cell line or the original cell lineage identity characteristics are lost after the primary cell line is established. (2) Most existing imaging technologies are targeted at marking repetitive sequences in the genome and are not suitable for DNA imaging of non-repetitive sequences. In the genomic DNA of eukaryotic cells, the promoter and enhancer regions related to gene expression regulation are mainly non-repetitive sequence DNA; the chromatin loop anchor region of the three-dimensional genome spatial conformation is also mainly non-repetitive sequence DNA. (3) Most existing imaging technologies are based on fixed cells and labeling genomic DNA for imaging. Only by labeling living cells can the spatial conformation of genomic DNA be tracked in situ, in real time, and dynamically. (4) More importantly, to analyze the causal relationship between the spatial structure of genomic DNA and gene expression regulation and cell fate, it is necessary to simultaneously label the anchor points of the genomic DNA spatial structure and the newly transcribed RNA of the related regions to establish the causal relationship between the high-dimensional structure of the genome and gene expression regulation.

[0006] Therefore, it is essential to develop methods suitable for primary cultured tissue cells, targeting non-repetitive DNA sequences, and dynamically tracking the spatial conformation of the genome and nascent transcripts in situ, in real time, and in living cells. Simultaneous imaging of DNA and RNA will reveal the causal relationship between the high-dimensional spatial conformation of the human genome and gene transcription and cell fate, providing a powerful tool for understanding the relationship between changes in genomic spatial structure, abnormal gene expression, and cell fate transitions during the development and progression of human disease.

[0007] Summary of the Invention

[0008] In view of the above-mentioned needs in the art, the present disclosure provides a construct comprising, in order from upstream to downstream, a structure represented by formula (I):

[0009] Promoter-tissue or cell-specific transcriptional regulatory element-dCas9 coding sequence-ribosomal skipping element-

[0010] dCas13 coding sequence-polyA

[0011] Formula (I).

[0012] In some embodiments, the promoter is a eukaryotic universal promoter. In some specific embodiments, the promoter is selected from any one of the following: CAG, CMV, EF1a.

[0013] In some embodiments, the tissue- or cell-specific transcriptional regulatory element is a Loxp-Stop-Loxp element.

[0014] In some embodiments, the coding sequence of dCas9 is optionally linked to a first tag element immediately upstream or downstream thereof.

[0015] In some embodiments, the coding sequence of dCas13 is optionally linked to a second tag element immediately upstream or downstream thereof.

[0016] In some embodiments, dCas13 is dCas13b or dCas13d.

[0017] In some embodiments, the tag element is selected from any one of the following: FLAG, HA, His, Myc.

[0018] In some embodiments, the first tag element and the second tag element are different.

[0019] In some embodiments, the ribosomal skipping element is selected from any one of the following: P2A, F2A, T2A, E2A, F2A30.

[0020] In some embodiments, the dCas9 is a Cas9 with a nuclease domain inactivating mutation. In some specific embodiments, the dCas9 is selected from any one of the following: SpCas9, NmCas9, Nme2Cas9, CjCas9, St1Cas9, SaCas9; preferably SpCas9.

[0021] In some embodiments, the coding sequence of dCas9 and the coding sequence of dCas13 can be interchanged.

[0022] In some embodiments, the dCas13d refers to Cas13d with an inactivating mutation in the HEPN domain.

[0023] In some embodiments, the dCas13b refers to Cas13b with a HEPN domain inactivating mutation.

[0024] In some embodiments, the construct further comprises a nuclear localization signal.

[0025] In some embodiments of the construct, the promoter is represented by SEQ ID NO: 1.

[0026] In some embodiments of the construct, the Loxp-Stop-Loxp element is represented by SEQ ID NO:2.

[0027] In some embodiments of the construct, the first tag element is set forth in SEQ ID NO:3.

[0028] In some embodiments of the construct, the coding sequence of dCas9 is shown in SEQ ID NO: 4.

[0029] In some embodiments of the construct, the ribosomal skipping element is set forth in SEQ ID NO:5.

[0030] In some embodiments of the construct, the coding sequence of dCas13d is shown in SEQ ID NO: 6.

[0031] In some embodiments of the construct, the second tag element is set forth in SEQ ID NO:7.

[0032] In some embodiments of the construct, the polyA is represented by SEQ ID NO:8.

[0033] The present disclosure also provides an expression vector comprising the aforementioned construct of the present disclosure.

[0034] In some embodiments, the expression vector is a eukaryotic expression vector.

[0035] In some specific embodiments, the expression vector is selected from any one of the following: an adenovirus expression vector, an adeno-associated virus expression vector, a lentivirus expression vector, a retrovirus expression vector, and a non-viral expression vector.

[0036] The present disclosure also provides use of the aforementioned construct or the aforementioned expression vector for preparing a transgenic animal model.

[0037] In some embodiments, the animal is a non-human animal.

[0038] In some embodiments, the non-human animal is selected from any one of the following: mouse, rat, guinea pig, rabbit, dog, primate, cow, sheep, camel, fruit fly, nematode, zebrafish, bird.

[0039] The present disclosure also provides use of the aforementioned construct or the aforementioned expression vector for preparing a cell line.

[0040] In some embodiments, the cell line is incapable of developing into a whole animal.

[0041] In some embodiments, the cell line is derived from any one of the following: mammals, insects, nematodes, birds, fish.

[0042] In some embodiments, the mammal is selected from any one of the following: human, mouse, rat, guinea pig, rabbit, dog, monkey, ape, cow, sheep, camel.

[0043] In some embodiments, the cell line is selected from any one of the following: embryonic stem cells, mesenchymal stem cells, fibroblasts, adult cells, tumor cells.

[0044] The present disclosure also provides use of the aforementioned construct or the aforementioned expression vector for real-time imaging of DNA and / or RNA in living cells.

[0045] In some embodiments, the living cells are from an animal model obtained according to the disclosed methods or uses.

[0046] In some embodiments, the living cells are ex vivo or in vitro.

[0047] In some embodiments, the real-time DNA imaging is selected from any one or a combination of the following: tracking of chromatin loops, tracking of chromatin interaction hubs, single-site genomic DNA imaging, multi-site genomic DNA imaging, genomic DNA imaging of repetitive sequence regions, and genomic DNA imaging of non-repetitive sequence regions.

[0048] In some embodiments, the real-time RNA imaging is selected from any one or a combination of the following: imaging of mRNA, subcellular localization of mRNA, imaging of non-coding RNA, subcellular localization of non-coding RNA, and tracking of transcripts.

[0049] In some embodiments, the DNA real-time imaging and the RNA real-time imaging are performed simultaneously or separately.

[0050] In some embodiments, the tracing of the transcript is selected from any one or a combination of the following: tracing of nascent transcription products, subcellular localization of mature RNA, and dynamic changes in nucleus / cytoplasm shuttling.

[0051] The present disclosure also provides a method for performing real-time DNA and / or RNA imaging of living cells in vitro, comprising the steps of:

[0052] 1) Providing living cells, wherein the living cells carry the aforementioned construct, or carry the aforementioned expression vector, or are isolated from the aforementioned animal model;

[0053] 2) providing the living cells with a Cre enzyme; preferably, providing the living cells with an expression vector for the Cre enzyme;

[0054] 3) providing the cells obtained in step 2) with a colorimetric marker-gRNA capable of binding to dCas9 or dCas13d, wherein the gRNA targets the site of interest;

[0055] 4) Observe the cells obtained in step 3) in real time under a microscope.

[0056] In some embodiments of the real-time imaging method, the DNA real-time imaging and the RNA real-time imaging are performed simultaneously or separately.

[0057] In some embodiments of the real-time imaging method, the site of interest is located in any one or a combination selected from the following: any repetitive sequence region in genomic DNA, any non-repetitive sequence region in genomic DNA, a chromatin loop anchor, a transcript of any gene of interest, or any non-coding RNA.

[0058] In some embodiments of the real-time imaging method, the colorimetric label is selected from any one or a combination of the following: a chromophore, a phosphorescent group, a photosensitizer, a chelating center, a heavy atom, a radioisotope, a magnetic particle, a benzoporphyrin, an azabenzoporphyrin, a naphthoporphyrin, a phthalocyanine, a polycyclic aromatic hydrocarbon, an azo dye, a xanthene dye, a boron dipyrromethene, an aza-boron dipyrromethene, a cyanine dye, a metal-ligand complex, an acridine, an aza -annulene, squarylium cyanine, 8-hydroxyquinoline, polymethine, quinolone, terbium complex, inorganic phosphor, ion carrier, Pd(II) octaethylporphyrin, Pd(II) tetraphenylporphyrin, Pt(II) meso-tetraphenylmethylbenzoporphyrin, Pt(II) octaethylporphyrin ketone, Pt(II) meso-tetrakis(pentafluorophenyl)porphyrin, Ru(II) tris(4,7-diphenyl-1,10-phenanthroline), Ru(II) tris(1,10-phenanthroline), tris(2,2″-bipyridyl) ruthenium(II) chloride hexahydrate, erythrosine B, fluorescein isothiocyanate, eosin, iridium(III)((N-methyl-benzimidazol-2-yl)-7-(diethylamino)-coumarin), indium(III)((benzothiazol-2-yl)-7-(diethylamino)-coumarin)-2-(acetylacetonate), Lumogen dyes, Macroflex fluorescent red, Macrolex fluorescent yellow, Texas Red, Rhodamine B, Rhodamine 6G, sulforhodamine, m-cresol, thymol blue, xylenol blue, cresol red, chlorophenol blue, bromocresol green, bromocresol red, bromothymol blue, Cy2, Cy3, Cy5, Cy5. 5. Cy7, 4-nitrophenol, alizarin, phenolphthalein, o-cresolphthalein, chlorophenol red, calcein, bromodimethylphenol, phenol red, neutral red, nitrazine, 3,4,5,6-tetrabromophenolphthalein, Congo red, 2", 7"-dichlorofluorescein, 5(6)-carboxyfluorescein, carboxynaphthofluorescein, 8-hydroxystyrene-1,3,6-trisulfonic acid, semi-naphthodifluorescein, semi-naphthofluorescein, tris(4,7-diphenyl-1,10-phenanthroline)dichlororuthenium(II), (4,7-diphenyl-1,10-phenanthroline)ruthenium(II)tetraphenylboron, dialkylcarbocyanine, dioctadecylcyclodicarbocyanine, fluorenylmethoxycarbonyl chloride, 7-amino-4-methylcoumarin, green fluorescent protein, yellow fluorescent protein, blue fluorescent protein, red fluorescent protein.

[0059] In some embodiments of the real-time imaging method, the non-coding RNA is selected from any one or a combination of the following: tRNA, rRNA, snoRNA, snRNA, TERC, tRF, tiRNA; miRNA, siRNA, piRNA, eRNA, lncRNA, circRNA, YRNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 Design scheme of DRIT transgenic mice, showing the names of sequence elements inserted into the genome.

[0061] Fig. 2 Genotype identification results of DRIT transgenic mice.

[0062] FIG3A shows labeling of the Akap6 gene locus in primary skeletal muscle stem cells, with targeting of a non-mouse genome-bound Gal4 gene as a negative control.

[0063] Figure 3B shows primary skeletal muscle cells from DRIT mice infected with the Ad-Cre-EGFP virus and imaged for Akap6 gene markers. Akap6 DNA marker signals were detected in EGFP-positive primary cells, while no DNA marker signals were detected in EGFP-negative cells.

[0064] Figure 3C shows representative fields of view showing successful labeling of one or a pair of Akap6 sites in skeletal muscle stem cells.

[0065] FIG3D compares the labeling efficiency of the disclosed method with that of fRNP in vitro assembly and delivery technology. The comparison index is the percentage of cells labeled to one or a pair of sites.

[0066] Figure 4 shows DNA labeling of three primary cells using the disclosed method. Primary skeletal muscle stem cells, primary fibroblasts, and primary preadipocytes were isolated from DRIT transgenic mice and infected with Cre virus. Fluorescent gRNA targeting the Akap6 gene locus was delivered to label the Akap6 gene locus.

[0067] FIG5A : The disclosed method labels non-repetitive genomic DNA. In primary skeletal muscle stem cells, a pool of gRNA fluorescent probes targeting the Myog and Mybph chromatin loop anchors was delivered by electroporation. Representative images of individual anchor labeling are shown.

[0068] FIG5B shows the statistical results of the positive rate of Myog and Mybph chromatin loop anchor non-repetitive sequences marked by the method disclosed herein.

[0069] Figure 5C: Representative images of simultaneous electroporation delivery of gRNA fluorescent probe pools targeting the Myog and Mybph chromatin loop anchors in primary skeletal muscle stem cells, showing simultaneous labeling of both anchors. Representative images 120 hours after delivery of the gRNA fluorescent probe pools.

[0070] FIG5D shows the statistical results of the positive ratio of two anchor points marked simultaneously by the method disclosed in the present invention.

[0071] Figure 6A is a flow chart illustrating the disclosed method for labeling RNA. Primary cells obtained by the disclosed method are isolated and infected with Cre to activate expression of the corresponding imaging element. Labeling of exogenous gene transcripts is accomplished by delivering a plasmid containing 24× the MS2 exogenous gene and a fluorescent gRNA that targets the MS2 gene transcript.

[0072] Figure 6B shows representative images of RNA imaging achieved by delivering 24×MS2 plasmids and gRNA targeting the MS2 gene to primary skeletal muscle stem cells obtained by the method of the present disclosure. No 24×MS2 plasmid was delivered as a negative control.

[0073] FIG6C is a statistical analysis of the relative fluorescence intensity of RNA in FIG6B .

[0074] FIG7A is a representative picture of simultaneous imaging of DNA and RNA by delivering gRNA targeting the Akap6 gene locus DNA and its transcripts into primary skeletal muscle stem cells obtained by the method disclosed in the present invention and inducing the cells to enter the differentiation period.

[0075] FIG7B is a bar graph showing the expression levels of the Akap6 gene during the proliferation and differentiation stages of skeletal muscle stem cells; the experimental data are derived from RNA sequencing results, with the unit being FPKM.

[0076] FIG7C is a statistical analysis of the fluorescence intensity of cellular RNA signals at two time periods in FIG7A .

[0077] FIG7D is a statistical analysis of the signal-to-noise ratio of DNA imaging mediated by the method of the present disclosure, where S / B represents the signal-to-noise ratio.

[0078] FIG7E is a statistical analysis of the signal-to-noise ratio of RNA imaging mediated by the method of the present disclosure, where S / B represents the signal-to-noise ratio.

[0079] FIG7F is a 2D distance analysis graph of the relative fluorescence intensity of DNA in the differentiation stage image in FIG7A .

[0080] FIG7G is a 2D distance analysis diagram of the relative fluorescence intensity of RNA in the differentiation stage images in FIG7A .

[0081] FIG8A shows anchor point distance statistics for live cell imaging of Myog-Mybph chromatin loop anchor points in myoblasts (i.e., skeletal muscle stem cells) during their proliferation and differentiation phases using the disclosed method. The images were taken with a 0.5-minute interval and a total duration of 9.5 minutes.

[0082] FIG8B shows the average value of the dynamic distance change of the Myog-Mybph chromatin loop anchor points observed in the two periods in live cell imaging in FIG8A .

[0083] FIG8C is a live cell imaging dynamic distance statistics diagram of the Myog-Mybph chromatin loop anchor points observed in the two stages of FIG8A .

[0084] FIG9A uses the disclosed method to mark the Myog gene loci and nascent transcripts during the proliferation and differentiation phases (1 hour of induction), wherein red represents the Myog gene loci and sky blue represents the Myog nascent transcripts.

[0085] FIG9B is the statistical analysis result of the fluorescence intensity of RNA markers in the two groups of cells in FIG9A .

[0086] FIG9C shows the statistics of the positive rate of newly expressed transcripts in the differentiation period (1 h of induction) marked by the method disclosed in the present invention.

[0087] FIG9D is a representative image of 3D reconstruction and trajectory simulation of the differentiation period (1 h of induction) marked using the disclosed method, showing both nascent transcript signals and signals that do not overlap with the Myog gene locus.

[0088] FIG9E is a statistical graph showing the total fluorescence values ​​of the two sites shown in FIG9D changing with time.

[0089] FIG9F is a statistical analysis of the total fluorescence values ​​of the two sites shown in FIG9D . DETAILED DESCRIPTION

[0090] The present disclosure is further described below through specific examples, but it should be understood that these examples are only for illustrating the present disclosure and are not intended to limit the scope of the present disclosure in any form.

[0091] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the presently disclosed subject matter belongs.

[0092] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties disclosed herein.

[0093] As used herein, the term "about" when referring to a value or amount of mass, weight, time, volume, concentration, or percentage is intended to include variations in the specified amount, which in some embodiments are ±10%, in some embodiments are ±5%, in some embodiments are ±1%, in some embodiments are ±0.5%, and in some embodiments are ±0.1%. It should also be understood that each point value between two specific numerical ranges is also disclosed. For example, if 10 to 15 is disclosed, then integers and decimals between 10 and 15, such as 11, 12, 13, and 14, are also disclosed.

[0094] Construct

[0095] The construct according to the present disclosure comprises, in order from upstream to downstream, the structure represented by formula (I):

[0096] Promoter-tissue or cell-specific transcriptional regulatory element-dCas9 coding sequence-ribosomal skipping element-

[0097] dCas13 coding sequence-polyA

[0098] Formula (I).

[0099] The constructs disclosed herein are applicable to eukaryotic animals, and therefore any universal eukaryotic promoter is suitable. A promoter is a sequence upstream of the transcription start site of a transcribed gene, serving as a location for regulatory elements and factors to bind, thereby facilitating RNA transcription. Examples of useful universal eukaryotic promoters include, but are not limited to, CAG, CMV, and EF1a.

[0100] Tissue-specific or cell-specific transcriptional regulatory elements enable the control and regulation of transcriptional events in specific cell or tissue types (e.g., intestinal, lung, neural, etc.) as needed. Tissue-specific or cell-specific transcriptional regulatory elements themselves are routinely available in the art. In the disclosed constructs, a preferred tissue- or cell-specific transcriptional regulatory element is a Loxp-Stop-Loxp element.

[0101] The LoxP-STOP-LoxP element consists of two LoxP elements at either end and a terminator in the middle. In the presence of the recombinase Cre, the two LoxP elements and the terminator in between are cleaved, allowing transcription of the downstream gene. In the absence of Cre, the terminator in the middle of the LoxP elements prematurely terminates gene transcription, preventing transcription of the downstream gene. Thus, the LoxP-STOP-LoxP element acts as a switch to control gene transcription.

[0102] For example, a LoxP-STOP-LoxP element can be used in a viral vector or other eukaryotic expression vector, and the LoxP-STOP-LoxP cassette can be introduced into a transgenic or knock-in animal and linked to a promoter. Alternatively, two transgenic or knock-in animals can be mated so that their offspring all have the LoxP-STOP-LoxP cassette (from parent 1) and Cre in their cells to excise the stop sequence in a subset of those cells (from parent 2), thereby generating the expression pattern of the heterologous gene in the offspring.

[0103] The downstream genes regulated by the LoxP-STOP-LoxP element are dCas9 and dCas13. Therefore, the positions of dCas9 and dCas13 can be interchanged without affecting their expression.

[0104] If expression needs to be identified or purified, dCas9 and dCas13d can be provided with different tag elements (such as a first tag element and a second tag element). In some embodiments, the coding sequence of the dCas9 is optionally connected to a first tag element in its immediate upstream or downstream. In some embodiments, the coding sequence of the dCas13 is optionally connected to a second tag element in its immediate upstream or downstream.

[0105] In some embodiments, the construct of the present disclosure comprises, in order from upstream to downstream, the structure represented by formula (I-1):

[0106] Formula (I-1): promoter-tissue or cell-specific transcriptional regulatory element-first tag element-dCas9 coding sequence-ribosomal jumping element-dCas13 coding sequence-second tag element-polyA.

[0107] In some embodiments, the construct of the present disclosure comprises, in order from upstream to downstream, the structure represented by formula (I-2):

[0108] Formula (I-2): promoter-tissue or cell-specific transcriptional regulatory element-dCas9 coding sequence-first tag element-ribosomal jumping element-dCas13 coding sequence-second tag element-polyA.

[0109] In some embodiments, the construct of the present disclosure comprises, in order from upstream to downstream, the structure represented by formula (I-3):

[0110] Formula (I-3): promoter-tissue or cell-specific transcriptional regulatory element-first tag element-dCas9 coding sequence-ribosomal jumping element-second tag element-dCas13 coding sequence-polyA.

[0111] In some embodiments, the construct of the present disclosure comprises, in order from upstream to downstream, the structure represented by formula (I-4):

[0112] Formula (I-4): promoter-tissue or cell-specific transcriptional regulatory element-dCas9 coding sequence-first tag element-ribosomal jumping element-second tag element-dCas13 coding sequence-polyA.

[0113] The tag element is not limited to a specific type, as long as it does not affect the expression / activity of dCas9 and dCas13. Commonly used tag elements in the art include, but are not limited to, FLAG, HA, His, and Myc. As an example, one downstream gene carries FLAG and the other downstream gene carries HA.

[0114] The dCas9 refers to a Cas9 with a nuclease domain inactivation mutation. A nuclease-inactivated Cas9 protein is referred to as dCas9. In some embodiments, dCas9 has an inactive (e.g., inactivated) DNA cleavage domain.

[0115] In some embodiments, a Cas9 variant with a mutation is provided that causes Cas9 to lose its catalytic activity but does not affect its ability to bind to the target DNA sequence. For example, such mutations include substitutions within the Cas9 nuclease domain (for example, but not limited to substitutions in the HNH nuclease subdomain, or the RuvC1 subdomain). In some embodiments, dCas9 can be obtained by existing technology, for example, but not limited to any one selected from the following: SpCas9, NmCas9, Nme2Cas9, CjCas9, St1Cas9, SaCas9. In some embodiments, dCas9 corresponds to or comprises some or all of the amino acids listed in SEQ ID NO: 4.

[0116] Cas13 is a Class 2 type VI RNA endonuclease with two HEPN domains for RNA cleavage. After Cas13 binds to the target RNA, the two HEPN domains undergo conformational changes, forming catalytic sites on the protein surface that randomly degrade target and non-target RNA. The Cas13 family includes Cas13a, Cas13b, Cas13c, Cas13d, Cas13x, Cas13y, etc. As an example, RNA imaging tools based on the CRISPR-Cas13 system in the prior art include:

[0117] - A fusion protein based on dLwaCas13a-msfGFP, which also includes a nuclear localization sequence, a zinc finger protein and a KRAB domain;

[0118] -dPspCas13b and dPguCas13b can be used for dual-channel imaging;

[0119] When dCas13-crRNA RNP is delivered, crRNA is protected by proteins, slowing its degradation reaction and significantly extending its half-life. This property, combined with dCas9, enables simultaneous imaging of the genome and transcriptome, a tool called CRISPR LiveFISH.

[0120] In some embodiments, dCas13d and dCas13b refer to Cas13d and Cas13b with HEPN domain inactivation mutations. dCas13 is obtained by mutating the R-XXXX-H motif in the HEPN domain of the Cas13 protein. The dCas13 protein loses its cleavage enzyme activity but retains its binding enzyme activity, thus enabling it to target target RNA molecules.

[0121] The coding sequences of dCas9 and dCas13 are separated by a ribosomal skipping element.

[0122] Ribosomal skipping is a unique mechanism of translation in which specific peptides prevent the ribosome from covalently attaching a newly inserted amino acid and allowing it to continue translation. Ribosomal skipping occurs both in vitro and in vivo during translation of eukaryotic ribosomes. Ribosomal skipping elements allow the production of proteins encoded by the element's flanking sequences. These flanking sequences prevent the ribosome from covalently attaching to the newly inserted amino acid and allow it to continue translation, resulting in co-translational cleavage of the polyprotein. Examples of ribosome skipping elements include, but are not limited to, P2A, F2A, T2A, E2A, and F2A30.

[0123] As an example, P2A is a small, self-cleaving peptide consisting of 19 amino acid residues: ATNFSLLKQAGDVEENPGP. This peptide doesn't actually self-cleave, but rather allows the ribosome to skip the synthesis of the glycine and proline peptide bonds at the C-terminus of the element, ultimately leading to the separation of the 2A end from the downstream product. Proteins expressed from upstream genes will have additional P2A residues (GSGATNFSLLKQAGD VEENPG) at their C-termini, while proteins expressed from downstream genes will have additional prolines at their N-termini. Adding a GSG sequence to the N-terminus of the P2A peptide improves cleavage efficiency.

[0124] Eukaryotic expression vector

[0125] The present disclosure also provides an expression vector comprising the construct represented by the aforementioned formula (I) of the present disclosure.

[0126] An expression vector is a vector that incorporates expression elements (such as promoters and terminators) into its basic backbone, enabling the replication, transcription, and translation of inserted foreign DNA / RNA within host cells. Depending on their function and purpose, expression vectors can be generally categorized as prokaryotic and eukaryotic.

[0127] The constructs disclosed herein can be inserted into any appropriate eukaryotic expression vector. Eukaryotic expression vectors can be divided into viral vectors and non-viral vectors.

[0128] Eukaryotic expression vectors are available in the prior art. Common viral expression vectors include, but are not limited to, adenoviral expression vectors, adeno-associated viral expression vectors, lentiviral expression vectors, and retroviral expression vectors.

[0129] Other common eukaryotic cell expression vectors include but are not limited to CMV4, pCMVp-NEO-BAN, pEGFP, pEGFP-N3, pEGFP-Actin, pSV2, pcDNA3.1, pPIC3.5, pPIC9K, pPIC Zα, pGAPZ A, pYC119, pYES2-CT, pAUR123, and pUC19DNA.

[0130] use

[0131] The present disclosure also provides the use of the aforementioned construct or expression vector for preparing a transgenic animal model. The present disclosure also provides the use of the aforementioned construct or expression vector for preparing a cell line that cannot develop into a complete animal individual.

[0132] Transgenic animals are animals that have foreign genes integrated into their chromosomal genomes. The foreign genes integrated into the animal's chromosomal genome are called transgenes. It should be understood that when creating transgenic animals, the foreign genes may be integrated into the genomes of only a portion of the animal's tissue cells or into the genomes of all of the animal's tissue cells. Animals with foreign genes integrated into the genomes of only a portion of their tissue cells are called chimeric animals. In such animals, the transgene is only heritable if the "partial tissue cells" into which the foreign genes are integrated are germ cells; otherwise, the foreign genes cannot be passed on to offspring.

[0133] As an example, the transgenic animal is a mouse, such as an SPF grade, which is not limited to a strain, such as but not limited to 129 / SvEv, C57BL / 6N, C57BL / 6J-HPRT, BALB / cAnN, CBA / CaO1a, 129 / SvJ, DBA / 2N, DBA / 101a, C3H / HeN, C57BL / 601a, FVB / N and Swiss Webster.

[0134] Any suitable method for preparing transgenic animal models or cell lines known in the art can be used. Depending on the method and target of exogenous gene introduction, methods include microinjection, retrovirus, embryonic stem cell, electric pulse, and sperm vector introduction.

[0135] As an example, the basic principle and method of microinjection is to inject the modified target gene into the fertilized egg or preimplantation embryonic cell of the donor mouse by microinjection, and then implant the fertilized egg or preimplantation embryonic cell into the oviduct or uterus of the recipient animal to develop it into a transgenic animal carrying the exogenous gene.

[0136] As another example, the electric pulse method, also known as electroporation, is to thoroughly mix the donor DNA with the recipient cells, and change the cell membrane structure under external high-voltage short pulses, causing instantaneous reversible electroporation of the cell membrane, so that DNA of a certain size can pass through the cell membrane into the cell and be transported to the cell nucleus.

[0137] Real-time imaging methods for DNA and / or RNA

[0138] The aforementioned construct or expression vector disclosed herein is also used for real-time imaging of DNA and / or RNA in living cells, wherein the living cells are isolated or in vitro and carry the aforementioned construct or expression vector.

[0139] In the imaging uses and methods disclosed herein, a complex formed by a color-labeled gRNA and a dCas protein can track genomic DNA in cells (including primary cells) and image RNA in cells. The present disclosure uses dCas9 and dCas13 to enable real-time imaging of genomic DNA and RNA transcripts in the same cell.

[0140] The gRNA typically comprises a target segment, wherein the target segment has complete or partial complementarity with the site of interest (nucleic acid sequence of interest).

[0141] In some embodiments, the gRNA molecules used in the present disclosure can vary in sequence length. The portion of the gRNA that is complementary to the site of interest, i.e., the targeting portion, can also vary in length and can be selected so that the gRNA can target the nucleic acid sequence of interest (target sequence, target site, etc.). In some embodiments, the length of the gRNA targeting portion is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, In some preferred embodiments, the targeting portion of the gRNA is about 15-25 nucleic acids in length. In some embodiments, the gRNA has a Cas binding portion other than the targeting portion. In some embodiments, the Cas binding portion is about 80 to about 100 nucleic acids in length, preferably about 90 nucleic acids in length.

[0142] Methods and tools for designing gRNAs are known to those of ordinary skill in the art. For example, crispr.mit.edu / , https: / / chopchop.rc.fas.harvard.edu / , www.e-crisp.org / , crispr.cos.uni-heidelberg.de / .

[0143] The colorimetric label carried by the gRNA is detectable, and its purpose is to trace the complex formed by the gRNA and the dCas protein. Therefore, all colorimetric labels that can be used for imaging in the prior art are applicable. Colorimetric labels include but are not limited to UV-Vis labels, near-infrared labels, luminescent groups, phosphorescent groups, magnetic spin resonance labels, photosensitizers, photocleavable parts, chelating centers, heavy atoms, radioisotopes, isotope detectable spin resonance labels, paramagnetic parts, chromophores, luminophores, such as metalloporphyrins; benzoporphyrins; azabenzoporphyrins; naphthoporphyrins, phthalocyanines; polycyclic aromatic hydrocarbons, such as perylene, perylene diimide, pyrene; azo dyes; xanthene dyes; boron dipyrromethene, aza-boron dipyrromethene, cyanine dyes, metal-ligand complexes such as bipyridine, bipyridyl , phenanthroline, coumarin, and acetylacetonates of ruthenium and iridium; acridine, oxazine derivatives such as benzophenoxazine; aza-annulene, squarylium cyanine; 8-hydroxyquinoline, polymethine, luminescence-producing nanoparticles such as quantum dots, nanocrystals; quinolones, terbium complexes; inorganic phosphors; ion carriers such as crown ethers attached or derivatized dyes; Pd(II)-octaethylporphyrin; Pt(II)-octaethylporphyrin; Pd(II) tetraphenylporphyrin; Pt(II) tetraphenylporphyrin; Pd(II) meso-tetraphenylporphyrin tetrabenzomorphine; Pt(II) meso-tetraphenylmethyl Benzoporphyrin; Pd(II) octaethylporphyrinone; Pt(II) octaethylporphyrinone; Pd(II) meso-tetrakis(pentafluorophenyl)porphyrin; Pt(II) meso-tetrakis(pentafluorophenyl)porphyrin; Ru(II) tris(4,7-diphenyl-1,10-phenanthroline) (Ru(dpp)3); Ru(II) tris(1,10-phenanthroline) (Ru(phen)3), tris(2,2"-bipyridyl)ruthenium(II) chloride hexahydrate (Ru(bpy)3); erythrosine B; fluorescein; fluorescein isothiocyanate (FITC); eosin; Iridium(III)((N-methyl-benzimidazol-2-yl)-7-(diethylamino)-coumarin); Indium(III)((benzothiazol-2-yl)-7-(diethylamino)-coumarin)-2-(acetylacetonate); Lumogen dyes; Macroflex fluorescent red; Macrolex fluorescent yellow; Texas Red; Rhodamine B; Rhodamine 6G; Sulforhodamine; m-cresol; Thymol blue; Xylenol blue; Cresol red; Chlorophenol blue; Bromocresol green; Bromocresol red; Bromothymol blue; Cy2; Cy3; Cy5; Cy5.5; Cy7; 4-nitrophenol; alizarin; phenolphthalein; o-cresolphthalein; chlorophenol red; calcein; bromodimethylol; phenol red; neutral red; nitrazine; 3,4,5,6-tetrabromophenolphthalein; Congo red; eosin; 2",7"-dichlorofluorescein; 5(6)-carboxy-fluorescein; carboxynaphthofluorescein; 8-hydroxystyrene-1,3,6-trisulfonic acid; semi-naphthodifluorescein; semi-naphthofluorescein; tris(4,7-diphenyl-1,10-phenanthroline) (4,7-Diphenyl-1,10-phenanthroline)ruthenium(II) dichloride; (4,7-Diphenyl-1,10-phenanthroline)ruthenium(II) tetraphenylboron; platinum(II) octaethylporphyrin; dialkylcarbocyanine; dioctadecylcyclodicarbocyanine; fluorenylmethoxycarbonyl chloride; 7-amino-4-methylcoumarin (Amc); green fluorescent proteins, for example, GFP, GFP-2, tagGFP, turboGFP, eGFP, Emerald, Azami Green, monomeric Azami Green, CopGFP, AceGFP, ZsGreenl, yellow fluorescent proteins (e.g., YFP, eYFP, Citrine, Venus, YPet, PhiYFP, ZsYellowl), blue fluorescent proteins (e.g., eBFP, eBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire), cyan fluorescent proteins (e.g., eCFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan), red fluorescent proteins (e.g., mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-monomer, HcRed-Tandem, HcRed1, AsRed2, eqFP611, mRaspberry, mStrawberry, Jred), orange fluorescent protein (e.g., mOrange, mKO, Kusabira-Orange, monomeric Kusabira-Orange, mTangerine, tdTomato), and any other suitable fluorescent protein.

[0144] There are many types of RNA in objects with complex functions. According to whether they encode proteins, they are divided into two major categories: coding RNA (mRNA) and non-coding RNA (ncRNA).

[0145] Non-coding RNAs can be divided into two categories based on their length: those longer than 200 nt are called long non-coding RNAs (lncRNAs), those shorter than 200 nt are called small non-coding RNAs (small ncRNAs), and those shorter than 50 nt are called tiny ncRNAs (such as siRNAs, miRNAs, and piRNAs). Non-coding RNAs can also be divided into housekeeping non-coding RNAs and regulatory non-coding RNAs based on their expression and functional properties. The constructs, expression vectors, and methods disclosed herein are suitable for imaging any type of coding and non-coding RNA.

[0146] Example

[0147] The novel imaging technology disclosed in the present invention is DRIT transgenic mice CAG-LSL-dCas9-P2A-dCas13d, with a genetic background of C57BL / 6J.

[0148] All animal experimental procedures were approved by the Experimental Animal Ethics Committee of the Institute of Basic Medicine, Chinese Academy of Medical Sciences (ACUC-A01-2022-017). All mice used in the examples were raised and bred in SPF-grade animal rooms. The feeding conditions were: 12 hours of light and dark alternation, the indoor temperature of the animal room was set at 19-24 ° C, and the humidity was 50%-60%. The feed, water, and bedding used by the mice were strictly sterilized at high temperature and high pressure, and all mice had free access to food and water.

[0149] The mouse genotype identification primers and imaging probe sequences involved in the present disclosure were designed using the Primer-BLAST tool and the CHOPCHOP tool, and were synthesized by Sangon Biotech (Shanghai) Co., Ltd. and IDT (Integrated DNA Technologies) of the United States.

[0150] Example 1: Design, preparation, and identification of constructs, transgenic animals, and cell lines

[0151] (1) Design ideas for transgenic animals

[0152] The method disclosed in the present invention is based on CRISPR gene editing technology, which utilizes the ability of the enzyme activity-deficient mutant dCas9 to target and bind to DNA under the guidance of RNA (i.e., gRNA), and the ability of the enzyme activity-deficient mutant dCas13 to target and bind to RNA under the guidance of gRNA, and achieves the targeting and fluorescent labeling tracking of specific DNA / RNA under the guidance of fluorescently modified gRNA (Fluo-gRNA).

[0153] The method disclosed herein is to add nuclear localization signals to elements such as dCas9, dCas13 (dCas13d or dCas13b) to prepare transgenic animals (such as mice), isolate and culture primary cells from the animals, and transfer fluo-gRNA into the cell nucleus by electroporation. Fluo-gRNA is assembled into a ribonucleic acid protein complex with dCas9 or dCas13 in the cell nucleus, targeting and identifying DNA or RNA to achieve simultaneous targeted labeling of DNA and RNA with repetitive or non-repetitive sequence characteristics in primary cells. The constructs and methods disclosed herein are suitable for primary cultured tissue cells, for repetitive or non-repetitive DNA sequences, and for in situ, real-time, and dynamic tracing of genome spatial conformation and nascent transcripts in living cells.

[0154] (2) Preparation of DRIT transgenic mice

[0155] Here, only mice are used as an example, and other species are not excluded. The mice constructed using the constructs and methods disclosed herein are hereinafter referred to as DRIT transgenic mice.

[0156] The inserted construct is CAG-LSL-dCas9-P2A-dCas13d (or dCas13b) (Figure 1). The sequence elements inserted into the genome include:

[0157] CAG: promoter sequence, used to initiate transgene expression;

[0158] LSL: Loxp-Stop-Loxp element, which enables tissue / cell-specific transgene expression;

[0159] dCas9: Cas9 with enzyme activity deletion mutant;

[0160] dCas13d (or dCas13b): Cas13d (or Cas13b) with enzyme activity deletion mutant;

[0161] P2A: enables the ribosome to skip the peptide bond synthesis at the C-terminus of the 2A element, separating the dCas9 and dCas13 proteins;

[0162] After the above design was completed, Jiangsu Jicui Pharmaceutical Co., Ltd. was commissioned to complete the preparation of transgenic mice.

[0163] (3) Methods for initiating expression of dCas9 and dCas13d (or dCas13b)

[0164] Method 1: Breeding of DRIT transgenic mice

[0165] The DRIT mice prepared by the present disclosure are mated with mice encoding tissue-specific Cre enzymes. The Cre enzyme recognizes the Loxp site and removes the Stop sequence, and the CAG promoter can then initiate the expression of dCas9 and dCas13d (or dCas13b). For example, DRIT mice are mated with mice (Pax7-Cre) that specifically express the Cre enzyme in skeletal muscle stem cells. The Stop sequence is specifically removed in the skeletal muscle stem cells of the offspring animals. Therefore, the CAG promoter specifically initiates the expression of dCas9 and dCas13d (or dCas13b) in skeletal muscle stem cells.

[0166] Method 2: If DRIT mice are not bred with Cre, primary cells of any tissue can be directly isolated from DRIT mice and infected with adenovirus encoding the Cre enzyme. The Cre enzyme removes the Stop sequence, and the CAG promoter drives the expression of dCas9 and dCas13d (or dCas13b) in the cells.

[0167] (4) Identification of DRIT transgenic mice

[0168] The toes or tails of unidentified mice were clipped and placed in a 1.5 mL tube. 200 μL of 50 mM NaOH solution was added and incubated in a 98°C water bath for 1.5-2 hours. After the incubation period, 20 μL of Tris-HCl (pH 8.0) was added and used as template DNA for PCR amplification. PCR primer sequences are shown in Table 1. PCR products were analyzed by 1.5% agarose gel electrophoresis. The PCR product in DRIT transgenic mice was 265 bp in size, while no amplified band was observed in wild-type control mice (Figure 2).

[0169] Conclusion: DRIT transgenic mice were successfully generated.

[0170] Example 2: DRIT for Genomic DNA Imaging of Primary Cultured Cells from Various Tissues

[0171] 1. Primary cell isolation and culture (taking mice as an example)

[0172] Isolation and culture of primary skeletal muscle stem cells and fibroblasts:

[0173] (1) Before isolating primary cells, all surgical instruments required were sterilized by dry heat; (2) Mice aged about 2-3 weeks (transgenic animals obtained from Example 1) were selected and killed by cervical dislocation. After surface disinfection with 75% alcohol, surgical scissors were used to cut the mouse abdominal cavity and extend to the foot joint of the lower limb to expose the lower limb muscles. All lower limb skeletal muscle groups were completely removed with surgical scissors and placed in PBS buffer; (3) The muscles were repeatedly washed in PBS and the fat and mouse hair attached to the muscles were removed; (4) The muscle tissue was transferred to a cell culture dish of corresponding size, and the muscle tissue was fully minced with surgical scissors. It was then digested with freshly prepared type II collagenase in a cell culture incubator at 37°C, and mixed by blowing every 15-20 minutes for a total of 40-60 minutes; (5) After the digestion was completed, the digestion was completed. Add an appropriate amount of PBS to the culture dish to terminate the digestion, filter the digestion solution and centrifuge at 400g for 8-10 minutes; (6) After the centrifugation, carefully discard the supernatant, add an appropriate amount of PBS to resuspend, and centrifuge again at 400g for 8-10 minutes; (7) After the centrifugation, discard the supernatant, add F10 culture medium to resuspend the precipitate, and transfer it to a new cell culture dish and culture it in a cell culture incubator for 30-40 minutes to purify skeletal muscle stem cells by differential adhesion; (8) After the differential adhesion is completed, transfer the non-adherent cells (supernatant) to a new cell culture dish that has been treated with collagen in advance, add FGF of the corresponding concentration and culture it; at the same time, the cells adhered in step (7) contain a large number of fibroblasts, which can be purified by further culture and differential adhesion in the future and cultured as fibroblasts.

[0174] Isolation and culture of primary fat precursor cells: (1) Before isolating primary cells, all surgical instruments required were sterilized by dry heat; (2) 2-3 week old mice were selected and killed by cervical dislocation. After surface disinfection with 75% alcohol, the abdominal cavity of the mice was cut open with surgical scissors, and the subcutaneous fat tissue of the inguinal region was exposed and peeled off and placed in PBS; (3) The obtained fat tissue was repeatedly washed in PBS and the mouse hair attached to the fat tissue was removed; (4) The fat tissue was transferred to a cell culture dish of corresponding size and placed in a surgical scissor. The adipose tissue was minced thoroughly and digested with freshly prepared type I collagenase in a 37°C cell culture incubator, mixing by pipetting every 15 minutes until digestion was complete; (5) After digestion was completed, an appropriate amount of PBS was added to the culture dish to terminate the digestion, the digestion solution was filtered, and centrifuged at 400g for 8-10 minutes; (6) After centrifugation, the supernatant was carefully discarded, and the precipitate was resuspended in F12 culture medium and transferred to a new cell culture dish for culture in a cell culture incubator for subsequent experimental use.

[0175] 2. Imaging Methods

[0176] Annealing and assembly of gRNA: Anneal equimolar amounts of crRNA and tracrRNA in annealing buffer. The annealing schedule is as follows: 95°C for 5 minutes; 70°C for 5 minutes; and 40°C for 5 minutes. After equilibration to room temperature, add MgCl2 to a final concentration of 1 mM. Mix well and incubate at 40°C for 5 minutes to complete RNA annealing.

[0177] Electroporation transfection: Nucleofector in Lonza electroporation kit TM The solution and supplement components were balanced to room temperature; the cells to be electroporated were removed, the supernatant was discarded and washed with PBS, and the cells were digested with trypsin and counted as 1×10 6 After centrifugation, discard all the supernatant as much as possible, add 100 μL of electroporation solution to resuspend the cell pellet, and transfer the cell suspension to the electroporation cup, avoiding the generation of bubbles as much as possible during the transfer process; select the electroporation program according to the cell type (such as CD-137 program for primary skeletal muscle stem cells); after the electroporation is completed, add 500 μL of cell culture medium to the electroporation cup, and use the matching pipette tip to aspirate the cell suspension in the electroporation cup and transfer it to a cell culture dish. After adding the corresponding cell culture medium, culture it in a cell culture incubator.

[0178] Live cell microscopy: transfer the cells to be imaged to a 35mm glass-bottom confocal dish suitable for super-resolution and live cell imaging, and observe them on a microscope equipped with a live cell imaging carrier. The live cell carrier must be preheated and its CO2 concentration adjusted to 5%. At the same time, inject an appropriate amount of sterile water to ensure moderate humidity in the carrier. Before imaging, use NucBlue TM Live ReadyProbes TM Live cell nuclear staining was performed; after staining, the culture medium was replaced with a phenol red-free FluoroBrite series medium and the 35mm glass-bottomed confocal microplate was transferred to a live cell imaging mount for observation. After adjusting the focus and fluorescence channels, the laser intensity and exposure time were further optimized. Observation in the near-UV channel was minimized or even eliminated, switching to brightfield or not observing at all to ensure sufficient cell viability for long-term live cell observation and avoid irreversible damage to cells caused by accumulated phototoxicity. For the actual imaging, the xyzt four-dimensional observation parameters were set to capture the 3D dynamic characteristics of the live cells within the field of view.

[0179] Image Processing and Calculation: Save the acquired 4D data and import them into Huygens and Imaris software for further processing. Once in Huygens, import the image to be processed and modify the excitation and emission spectra identified by the Huygens software according to the parameters used during capture to ensure accurate deconvolution. Select the Deconvolution function, select the channel to be deconvolved, and perform background estimation in manual mode. Set appropriate parameters such as Maximum Iterations, Signal to Noise Ratio, and Quality Threshold to perform the deconvolution setup. After deconvolution, perform automatic Z-drift correction and chromatic aberration (optional) and save the processed file to complete the entire process. Further processing of the raw data or deconvolved Huygens file using Imaris software involves 3D reconstruction of fluorescence within different channels to locate the 3D position of the fluorophores at each moment and calculate the 3D distances between them. The localization and dynamic trajectory of fluorescent groups in cells are displayed through 2D to 3D cell sections and path simulation reconstruction, respectively.

[0180] 3. Labeling and Imaging of Repetitive DNA

[0181] To verify the labeling ability of the disclosed method for DNA in primary cells, skeletal muscle stem cells were isolated from the skeletal muscle of DRIT mice, infected with adenovirus (Ad-Cre-EGFP) expressing Cre protein and EGFP protein, and activated the expression of dCas9 and dCas13d in skeletal muscle stem cells. For the target sequence in mice (taking the repeat sequence of chromosome 12 Akap6 gene as an example), a Cy5 fluorescently modified gRNA (see Table 2) that can target the Akap6 gene site was synthesized. The probe can bind to 87 consecutive copies at the Akap6 gene site. At the same time, a fluorescent gRNA targeting the Gal4 gene sequence specifically present in the Drosophila genome was synthesized as a negative control. By electroporation transfection, gRNA was delivered to primary skeletal muscle stem cells, targeted to the Akap6 gene repeat sequence region in the nucleus, and live cell observation was taken with a 63x oil lens under a DMi8 microscope.

[0182] The results showed that the method disclosed herein can effectively mark the repetitive sequence of Akap6 DNA, while targeting the Gal4 gene that does not exist in the mouse genome (negative control) will not produce a signal in the cell nucleus (Figure 3A). Moreover, we found that the Akap6 signal is only seen in cells infected with Ad-Cre-EGFP and activating the expression of dCas9 protein, while cells that are not infected with Cre will not be marked, proving that the constructs and methods disclosed herein have strong accuracy (Figures 3B to 3C). Compared with the DNA labeling method of assembling gRNA and delivering the dCas9 complex into the nucleus in vitro, the method disclosed herein is more efficient in labeling DNA in primary cells, with a positive rate of more than 50%, and is more prominent in the integrity of single cell (diploid) labeling (Figure 3D).

[0183] 4. DNA imaging in various primary cultured cells

[0184] To verify the compatibility of the disclosed method with various primary cells, DNA labeling imaging of target sequences (taking the Akap6 gene locus as an example) was tested in primary skeletal muscle stem cells, primary fibroblasts, and primary adipocytes.

[0185] It was found that the method disclosed herein can be used to achieve DNA labeling in different primary cells ( FIG. 4 ), which indicates that the method has a very wide range of applications.

[0186] 5. Labeling and Imaging of Non-repetitive DNA Sequences

[0187] In order to detect the efficiency of the method disclosed in the present invention for imaging non-repetitive sequences in the genome, the chromatin loop anchors formed by the Myog and Mybph gene sites were selected for labeling. 15 consecutive gRNA probes were designed within 5kb of the two anchor points, and fluorescent group-labeled gRNAs were synthesized to form two gRNA fluorescent probe pools. The gRNA was annealed and electroporated into primary skeletal muscle stem cells. After 24 hours, imaging was performed using a DMi8 inverted fluorescence microscope equipped with sCMOS with a 63x (NA = 1.40) oil objective or using a stimulated emission depletion (STED) super-high resolution microscope with a 100x oil objective.

[0188] The results showed that the use of a gRNA fluorescent probe pool composed of 15 fluorescently labeled gRNAs can achieve non-repetitive sequence DNA labeling in the chromatin loop anchor region, and the overall positive rate of each labeling can reach about 50% (Figure 5A to Figure 5B), and it is possible to label two anchors at the same time (Figure 5C to Figure 5D). More importantly, when the sample cells were observed after 120 hours, relatively bright labels could still be observed, which shows that the method disclosed herein uses a fluorescent probe pool composed of 15 gRNAs to label non-repetitive sequences. It is very stable (Figure 5C).

[0189] Conclusion: The disclosed method can be used to image genomic DNA of repetitive and non-repetitive sequences in primary cultured cells from a variety of tissues. "Primary" is not limited to initially isolated primary cells, but can also refer to cells cultured and expanded in vitro for an appropriate number of generations (e.g., but not limited to 1-5 generations). Primary cells from different tissues can be passaged at different times; for example, primary skeletal muscle cells can be passaged both in primary and in vitro for 2-3 generations.

[0190] Example 3: RNA imaging

[0191] To explore whether the method disclosed herein can complete the labeling of specific RNA by expressing dCas13d and introducing a specific targeted fluorescent gRNA probe into the cell, an MS2 plasmid with 24 continuous copies is used as an exogenous gene to detect the labeling ability of exogenous gene transcripts. First, primary skeletal muscle stem cells were isolated from mice prepared in Example 1, infected with adenovirus encoding Cre enzyme (Ad-Cre), and electroporated to transfect exogenous 24 × MS2 plasmids (4 μg) and fluorescent gRNA (50-80 pmo) containing dCas13d binding skeleton and targeting MS2 transcript sequence (Figure 6A). The empty plasmid transfection group was used as a negative control group, and imaging observation was performed after 24 hours of electroporation transfection.

[0192] The results showed that gRNA fluorescence inside and outside the nucleus could be clearly observed in cells transfected with the MS2 plasmid, while no gRNA fluorescence signal was accumulated in the negative control group (Figure 6B-Figure 6C). Because the number of copies of the plasmid taken up by different cells during transfection is different from that of the fluorescent gRNA, when performing statistics on the gRNA fluorescence signal of a single cell, there may be problems such as excessive background or excessive transcripts caused by more plasmids taken up, which will lead to larger fluctuations in the results. However, the labeling of the MS2 transcript can still be significantly seen by calculating the relative fluorescence value (Figure 6C). This proves that the method disclosed herein can label RNA.

[0193] Conclusion: The constructs and methods disclosed herein can achieve RNA labeling imaging.

[0194] Example 4: Simultaneous Imaging of Genomic DNA and RNA in Primary Cultured Cells

[0195] Using endogenous gene transcripts as targets, the accuracy of RNA imaging labeling was tested. While labeling the gene transcripts, the DNA at the gene locus was also labeled to verify the compatibility of the disclosed method for simultaneous imaging of DNA and RNA, further demonstrating the technology's application flexibility and accuracy.

[0196] The Akap6 gene, which is significantly upregulated during skeletal muscle stem cell differentiation, was selected for RNA imaging validation (Figure 7A). This gene has multiple consecutive copies in the intron (gRNA can target 87 of these consecutive copy sites). Fluorescent gRNA for RNA imaging was delivered to image the nascent transcripts of the Akap6 gene. A DNA imaging gRNA probe that can bind to dCas9 and target the Akap6 gene site was also introduced to validate the ability to simultaneously image both DNA and RNA.

[0197] First, skeletal muscle stem cells were isolated from the mice prepared in Example 1, infected with adenovirus encoding the Cre enzyme (Ad-Cre), and transfected with gRNA probes for DNA imaging and RNA imaging by electroporation. Live cell imaging observations were performed 24 hours later, and the imaging results of proliferating cells were recorded. Then, skeletal muscle stem cells were induced to differentiate and live cell imaging observations were performed.

[0198] The results showed that DNA labeling of the Akap6 gene locus can be observed in both the proliferation and differentiation phases, while RNA signals are only present and extremely significant during the differentiation phase (Figure 7B). After comparing the fluorescence intensity of the RNA signals in the two periods after background normalization, it was found that the trend of change was consistent with the results of RNA-seq, which proves that the method disclosed herein can achieve sensitive and accurate simultaneous labeling of DNA and RNA. Moreover, the labeled DNA and RNA signals can be collected by a fluorescence microscope with a relatively high signal-to-noise ratio (Figures 7C to 7G), further illustrating the superiority of the method disclosed herein in DNA and RNA imaging.

[0199] Conclusion: The constructs and methods disclosed herein can achieve simultaneous imaging of genomic DNA and RNA in primary cultured cells.

[0200] Example 5: Characterizing the dynamic characteristics of chromatin loops by imaging genomic DNA in living cells

[0201] Using the disclosed method, the dynamic characteristics of Myog and Mybph chromatin loops were investigated during the proliferation and differentiation phases of skeletal muscle stem cells (0.5 hours after changing the differentiation medium). The dynamic characteristics of Myog-Mybph chromatin loops were observed in living cells using a 30-second interval and a total observation time of 10 minutes.

[0202] The results showed that in skeletal muscle stem cells, the chromatin loop structure can be imaged by Z-stack in a manner compatible with live cell imaging (Figures 8A to 8B). And for the proliferation period of skeletal muscle stem cells, the corresponding chromatin loop anchor point distance has the characteristics of large fluctuation amplitude in a short period of time and long distance between anchor points, and basically meets the characteristics that the chromatin loop anchor point distance at each time point is greater than 150nm (Figure 8A). For the chromatin loop anchor point distance during the differentiation period of skeletal muscle stem cells, it has the characteristics of being relatively stable and its size is basically stable and less than 150nm (Figure 8A). Not only that, the average distance of the chromatin loop anchor point at each time point of live cell imaging of multiple different cells was further calculated, and it was found that the averaged result also met the characteristics of 150nm (Figures 8B to 8C), which is consistent with the fact that Myog-Mybph chromatin loops are only formed during the differentiation period of skeletal muscle stem cells.

[0203] Conclusion: The constructs and methods disclosed herein can be used to track chromatin looping in real time and dynamically in living cells.

[0204] Example 6: Labeling chromatin loops in living cells and RNA of newly transcribed genes

[0205] After demonstrating that the disclosed method can accurately label endogenous and exogenous gene transcripts, the characteristics of gene expression at chromatin loops were further explored.

[0206] Twenty fluorescent gRNA probes were designed and arranged serially within the Myog gene introns to label and track the nascent Myog gene transcripts. Skeletal muscle stem cells from mice prepared in Example 1 were isolated and infected with Ad-Cre virus. A pool of fluorescently modified gRNA probes targeting both Myog transcripts and Myog genomic DNA was then electroporated into the cells. Live cell imaging of the Myog gene locus and its nascent transcript RNA was performed 24 hours after electroporation using a DMi8 inverted fluorescence microscope equipped with a live cell imaging mount and sCMOS at a 63x (NA = 1.40) oil objective.

[0207] The results showed that the method disclosed herein can be used to simultaneously image DNA and RNA, and the Myog RNA signal observed after 1 hour of induction of differentiation is much higher than the RNA signal during the proliferation period, which is consistent with the specific induction of Myog expression during the differentiation period of skeletal muscle stem cells (Figures 9A to 9B). Moreover, the efficiency of labeling RNA using the method disclosed herein can reach 50% (Figure 9C). More importantly, by analyzing the dynamic changes in the fluorescence intensity of the Myog nascent transcript signal (overlapping with the labeled Myog DNA signal) and other intracellular site signals (miscellaneous signals or undegraded intron sequence signals) during the differentiation period, it was found that the fluorescent signal of the nascent transcript labeled by the method disclosed herein is very stable and more resistant to quenching. At the same time, as the Myog nascent transcript is continuously produced, the fluorescence intensity accumulated by the site labeling is enhanced as a whole compared to the photobleaching background fluorescence signal (Figures 9D to 9F).

[0208] Conclusion: The constructs and methods disclosed herein can stably and sensitively label genomic DNA and nascent transcript RNA simultaneously.

[0209] In summary, the method disclosed herein is different from the existing C-series technology in that it can directly observe the chromatin structure of a single living cell dynamically and analyze the chromatin structure at a 4D rather than 3D level, thus avoiding the bias in the interpretation of chromatin structure caused by the preferences of different C-series technologies. Compared with the labeling method of assembling the gRNA-dCas9 (dCas13) complex in vitro and delivering it into the cell nucleus, the dCas9 or dCas13 in the method disclosed herein is expressed in vivo / intracellularly, and only the gRNA with the targeting probe is delivered, so the labeling efficiency of DNA and RNA is higher.

[0210] Example sequence:

[0211] The CAG promoter sequence is (SEQ ID NO: 1):

[0212] The LSL sequence is (SEQ ID NO: 2):

[0213] The Flag sequence is (SEQ ID NO: 3):

[0214] The dCas9 sequence is (SEQ ID NO: 4):

[0215] The P2A sequence is (SEQ ID NO: 5):

[0216] The dCas13d sequence is (SEQ ID NO: 6):

[0217] The HA sequence is (SEQ ID NO: 7):

[0218] The polyA sequence is (SEQ ID NO: 8):

[0219] Table 1. Primer sequences for mouse genotyping

[0220] Table 2. Probe sequences used for genomic DNA and transcript RNA imaging

Claims

1. A construct comprising, in order from upstream to downstream, the structure represented by formula (I): Promoter-tissue or cell-specific transcriptional regulatory element-dCas9 coding sequence-ribosomal jumping element-dCas13 coding sequence-polyA Formula (I); in: The coding sequence of the dCas9 and the coding sequence of the dCas13 can be interchanged; The promoter is a eukaryotic universal promoter, which is selected from any one of the following: CAG, CMV, EF1a; The tissue or cell-specific transcriptional regulatory element is a Loxp-Stop-Loxp element; The coding sequence of dCas9 is optionally connected to a first tag element immediately upstream or downstream thereof; The coding sequence of dCas13 is optionally connected to a second tag element immediately upstream or downstream thereof; The dCas13 is dCas13d or dCas13b; The tag element is selected from any one of the following: FLAG, HA, His, Myc; the first tag element and the second tag element are different; The ribosomal skipping element is selected from any one of the following: P2A, F2A, T2A, E2A, F2A30; The dCas9 refers to a Cas9 with a nuclease domain inactivation mutation; it is selected from any one of the following: SpCas9, NmCas9, Nme2Cas9, CjCas9, St1Cas9, SaCas9; preferably SpCas9; The dCas13d refers to Cas13d with an inactivating mutation in the HEPN domain; The dCas13b refers to Cas13b with an inactivating mutation in the HEPN domain; Optionally, the construct further comprises a nuclear localization signal.

2. The construct according to claim 1, comprising any one or a combination thereof selected from the following: The promoter shown in SEQ ID NO: 1; The Loxp-Stop-Loxp element shown in SEQ ID NO: 2; The first tag element shown in SEQ ID NO: 3; The coding sequence of dCas9 shown in SEQ ID NO: 4; The ribosomal jumping element shown in SEQ ID NO: 5; The coding sequence of dCas13d shown in SEQ ID NO: 6; The second tag element shown in SEQ ID NO: 7; The polyA shown in SEQ ID NO:

8.

3. An expression vector comprising the construct according to claim 1 or 2; The expression vector is a eukaryotic expression vector; Preferably, the expression vector is selected from any one of the following: an adenovirus expression vector, an adeno-associated virus expression vector, a lentivirus expression vector, a retrovirus expression vector, and a non-viral expression vector.

4. Use of the construct according to claim 1 or 2 or the expression vector according to claim 3 for preparing a transgenic animal model, wherein: The animal is a non-human animal selected from any one of the following: mouse, rat, guinea pig, rabbit, dog, primate, cow, sheep, camel, fruit fly, nematode, zebra fish, bird.

5. Use of the construct according to claim 1 or 2 or the expression vector according to claim 3 for preparing a cell line, wherein: The cell line is incapable of developing into a complete animal individual; The cell line is derived from any of the following: mammals, insects, nematodes, birds, fish; The mammal is selected from any one of the following: human, mouse, rat, guinea pig, rabbit, dog, monkey, ape, cow, sheep, camel; Preferably, the cell line is selected from any one of the following: embryonic stem cells, mesenchymal stem cells, fibroblasts, adult cells, tumor cells.

6. Use of the construct according to claim 1 or 2 or the expression vector according to claim 3 for real-time imaging of DNA and / or RNA in living cells, wherein: The living cells are from the animal model defined in claim 4; The living cells are ex vivo or in vitro; The real-time DNA imaging is selected from any one of the following or a combination thereof: tracing of chromatin loops, tracing of chromatin interaction hubs, single-site genomic DNA imaging, multi-site genomic DNA imaging, genomic DNA imaging containing repetitive sequence regions, and genomic DNA imaging of non-repetitive sequence regions; The RNA real-time imaging is selected from any one or a combination of the following: mRNA imaging, mRNA subcellular localization, non-coding RNA imaging, non-coding RNA subcellular localization, and transcript tracking; The DNA real-time imaging and the RNA real-time imaging are performed simultaneously or separately; Preferably, the tracing of the transcript is selected from any one or a combination of the following: tracing of nascent transcription products, subcellular localization of mature RNA, and dynamic changes in nucleus / cytoplasm shuttling.

7. A method for real-time imaging of DNA and / or RNA in living cells in vitro, comprising the steps of: 1) Providing living cells, wherein the living cells carry the construct according to claim 1 or 2, or carry the expression vector according to claim 3, or are isolated from the animal model defined in claim 4; 2) providing the living cells with Cre enzyme; preferably, providing the living cells with an expression vector for the Cre enzyme; 3) providing the cells obtained in step 2) with a colorimetric marker-gRNA capable of binding to dCas9 or dCas13d, wherein the gRNA targets the site of interest; 4) observing the cells obtained in step 3) in real time under a microscope; in: The DNA real-time imaging and the RNA real-time imaging are performed simultaneously or separately; Preferably, the site of interest is located in any one or a combination selected from the following: a repetitive sequence region in genomic DNA, a non-repetitive sequence region in genomic DNA, a chromatin loop anchor, a transcript of a gene of interest, and a non-coding RNA.

8. The method according to claim 7, wherein the color marker is selected from any one of the following or a combination thereof: a chromophore, a phosphorescent group, a photosensitizer, a chelating center, a heavy atom, a radioactive isotope, a magnetic particle, a benzoporphyrin, an azabenzoporphyrin, a naphthoporphyrin, a phthalocyanine, a polycyclic aromatic hydrocarbon, an azo dye, a xanthene dye, a boron dipyrromethene, an aza-boron dipyrromethene, a cyanine dye, a metal-ligand complex, an acridine, an aza-annulene, a square acid cyanine, 8-hydroxyquinoline, a polymethine, a quinolone, a terbium complex, an inorganic phosphor, an ion carrier, Pd(II)-octaethylporphyrin, Pt(II)-octaethylporphyrin, Pd(II)-tetraphenyl Porphyrin, Pt(II) tetraphenylporphyrin, Pd(II) meso-tetraphenylporphyrin tetrabenzomorphine, Pt(II) meso-tetraphenylmethylbenzoporphyrin, Pd(II) octaethylporphyrinone, Pt(II) octaethylporphyrinone, Pd(II) meso-tetra(pentafluorophenyl)porphyrin, Pt(II) meso-tetra(pentafluorophenyl)porphyrin, Ru(II) tris(4,7-diphenyl-1,10-phenanthroline), Ru(II) tris(1,10-phenanthroline), tris(2,2"-bipyridyl) ruthenium(II) chloride hexahydrate, erythrosine B, fluorescein isothiocyanate, eosin, iridium(III) ((N-methyl- benzothiazol-2-yl)-7-(diethylamino)-coumarin), indium(III)((benzothiazol-2-yl)-7-(diethylamino)-coumarin)-2-(acetylacetonate), Lumogen dyes, Macroflex fluorescent red, Macrolex fluorescent yellow, Texas red, rhodamine B, rhodamine 6G, sulforhodamine, m-cresol, thymol blue, xylenol blue, cresol red, chlorophenol blue, bromocresol green, bromocresol red, bromothymol blue, Cy2, Cy3, Cy5, Cy5.5, Cy7, 4-nitrophenol, alizarin, phenolphthalein, o-cresolphthalein, chlorophenol red, calcein, bromo Xylenol, phenol red, neutral red, nitrazine, 3,4,5,6-tetrabromophenolphthalein, Congo red, 2", 7"-dichlorofluorescein, 5(6)-carboxy-fluorescein, carboxynaphthofluorescein, 8-hydroxystyrene-1,3,6-trisulfonic acid, semi-naphthodifluoride, semi-naphthofluorescein, tris(4,7-diphenyl-1,10-phenanthroline)dichlororuthenium(II), (4,7-diphenyl-1,10-phenanthroline)ruthenium(II)tetraphenylboron, dialkylcarbocyanine, dioctadecylcyclodicarbocyanine, fluorenylmethoxycarbonyl chloride, 7-amino-4-methylcoumarin, green fluorescent protein, yellow fluorescent protein, blue fluorescent protein, red fluorescent protein.

9. The method according to claim 7, wherein the non-coding RNA is selected from any one or a combination of the following: tRNA, rRNA, snoRNA, snRNA, TERC, tRF, tiRNA; miRNA, siRNA, piRNA, eRNA, lncRNA, circRNA, YRNA.

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