CN115305255A - Living cell ribosome RNA visualization system and application thereof - Google Patents

Abstract

The invention discloses a living cell ribosome RNA visualization system and application thereof. Specifically, the invention establishes a high-efficiency screening system for targeting rRNA generation, and the screening system is based on the newly developed living cell rRNA fluorescent reporter molecule. Compared with the traditional screening strategy, the system can greatly simplify the screening process and save the screening time, thereby remarkably improving the screening efficiency.

Description

Living cell ribosome RNA visualization system and application thereof

Technical Field

The invention belongs to the fields of molecular biology and biomedicine, and particularly relates to a ribosome RNA visualization system and application thereof.

Background

The key steps of ribosome production are carried out in the nucleolus, 47S pre-rRNA is produced by rDNA transcription, three kinds of ribosomal RNA, 18S, 5.8S and 28S respectively, are generated by shearing processing, are assembled into ribosome size subunits together with other components, and are transported to cytoplasm to finally form ribosome, which is used as a plant for protein synthesis. Ribosomes are involved in cell growth, proliferation, differentiation, cell life, embryonic development, neurodegeneration, and the development of cancer. The enlargement and number of nucleoli has long been a characteristic feature of tumor cells. This prominent feature is due to the over-activation of RNA polymerase I (Pol I), which generates large amounts of ribosomal RNA (rRNA), supporting the rapid growth and proliferation of tumor cells. The rate-limiting step in ribosome production is Pol I synthesis of rRNA.

Cancer has now become a common disease in modern society, but there is a constant search for how to treat cancer. A great deal of research has been conducted on cancer, and the understanding of cancer is continuously deepened, and at the same time, various methods for treating cancer, such as small molecule drugs, antibodies, and immunotherapy, have been developed. The nature of tumors is a multigenic mutated and complex disease, with high heterogeneity in molecular genetics and instability of its genome. As tumor cells continue to divide and proliferate, genomic mutations continue to accumulate. These characteristics pose major challenges and bottlenecks in tumor therapy. The targeted drug is one of the main directions of tumor therapy and drug development, and the treatment strategy has better curative effect and reduced side effect compared with the traditional chemotherapy drug. However, one of the major obstacles to cancer treatment is drug resistance. Tumor cells accumulate genetic mutations throughout the tumor as they divide and proliferate, allowing them to develop new escape mechanisms. Therefore, a targeted therapy strategy which is more lethal to tumor cells and more universal in targeting various tumor types is developed. Perhaps "non-personalized therapy" is the hope of tumor therapy. The strategy for targeted ribosome production can be said to be such a concept.

Regulation of ribosome biogenesis involves multiple steps, including transcription and processing of rRNA, synthesis of ribosomal proteins, and ribosome assembly, which are potential targets for inhibition of cell proliferation. Since most growth-promoting signals will be based on hyperactivated Pol I transcription, specific inhibition of its activity offers a particular therapeutic opportunity. Reducing ribosome production by inhibiting Pol I transcription may be an effective strategy for tumor therapy with several advantages: 1) Pol I mediates rRNA precursor transcription specifically in cells, i.e., the 47S rRNA precursor; 2) Abnormalities in ribosome production occur in most tumor cell types, and therefore Pol I transcription inhibitors have the potential to treat a variety of cancers; 3) Healthy somatic cells produce lower levels of ribosomes, and they have reduced sensitivity to Pol I inhibitors. It is therefore desirable to identify small molecules that selectively inhibit Pol I activity in tumor cells without affecting normal cells. Based on the research idea, three kinds of small anticancer molecular inhibitors including BMH-21, CX-5461 and CX-3543 have been identified by detecting the expression change of rRNA through a real-time fluorescent quantitative PCR or Northern blot hybridization mode. Where CX-5461 has entered clinical trials. However, the conventional real-time fluorescent quantitative PCR or Northern blot hybridization technique has complex screening process and long screening period, and is difficult to complete large-scale high-throughput screening.

Disclosure of Invention

The invention aims to provide a ribosomal RNA visualization system and application thereof.

In a first aspect of the invention, a genetically engineered cell is provided having incorporated into its rDNA an exogenous DNA sequence encoding an RNA marker sequence that becomes part of rRNA upon transcription of the rDNA. The RNA sequence contains several stem-loop repeats that bind multiple copies of the RNA-binding protein, thereby enriching each rRNA molecule for a sufficiently strong fluorescent signal for visual detection.

In another preferred example, the RNA marker sequence is MS2 sequence unit, PP7 sequence unit, and boxB sequence unit.

In another preferred embodiment, the insertion site of the foreign DNA at the rDNA is selected from the group consisting of: 5 ″ -ETS region, 18S region, ITS1 region, 5.8S region, ITS2 region, 28S region, or 3' ETS region; preferably 5'ETS region, ITS1 region, 5.8S region, or 3' ETS region; more preferably in the 5.8S region.

In another preferred embodiment, the RNA marker sequence comprises at least 6 RNA stem-loop repeats; preferably, at least 12 stem-loop repeats are included; more preferably, at least 17 stem-loop repeats are included.

In another preferred example, the MS2 sequence unit comprises at least 6 MS2 repeat sequences; preferably, at least 12 MS2 repeats are included; more preferably, at least 17 MS2 repeats are included.

In another preferred embodiment, the PP7 sequence unit comprises at least 6 PP7 repeat sequences; preferably, at least 12 PP7 repeats are included; more preferably, at least 17 PP7 repeats are included.

In another preferred example, the boxB sequence unit comprises at least 6 boxB repeated sequences; preferably, at least 12 boxB repeats are included; more preferably, at least 17 boxB repeats are included.

In another preferred embodiment, the genetically engineered cell further expresses an RNA binding protein that specifically binds to the RNA marker sequence.

In another preferred embodiment, the RNA binding protein is linked to a reporter molecule; preferably, the reporter molecule is a fluorescent protein or fluorescein.

In another preferred example, the fluorescent protein is a red fluorescent protein (e.g., tdTomato, dsRed, mCherry), a green fluorescent protein (e.g., eGFP, zsGreen), a Yellow Fluorescent Protein (YFP), a Blue Fluorescent Protein (BFP), a cyan fluorescent protein gene (CFP), etc.

In another preferred embodiment, when the RNA tag sequence is a MS2 sequence unit, the RNA binding protein is an MCP protein.

In another preferred embodiment, when the RNA tag sequence is a unit of PP7 sequence, the RNA binding protein is a PCP protein.

In another preferred embodiment, when the RNA marker sequence is a unit of a boxB sequence, the RNA binding protein is a lambda-N protein.

In another preferred embodiment, the cell is a mammalian cell, preferably a HEK293 cell, or a tumor cell such as Hela.

In another preferred embodiment, the genetically engineered cell is a monoclonal cell strain.

In a second aspect of the invention, there is provided a nucleic acid construct for gene knock-in, the nucleic acid construct having a structure represented by formula I or formula II,

S-M-S, formula I

S-5'HA-M-3' HA-S, formula II

In formula I, the S element is a target sequence which can be recognized and cut by the Cas9 protein, and the M element is a donor sequence; each "-" is a bond or a connecting sequence;

in formula II, the S element is a target sequence which can be recognized and cut by Cas9, and the M element is a donor sequence; 5'HA element is the homology arm 5' of the genomic target site into which the donor sequence is to be inserted, 3'HA element is the homology arm 3' of the genomic target site into which the donor sequence is to be inserted, each "-" being a bond or a linking sequence.

In another preferred embodiment, the sequence structure of the S element is as follows:

GCACCGATGCTCTCCGAGG(SEQ ID NO.11)。

in another preferred embodiment, the M element is a DNA fragment that is transcribed into an RNA marker sequence.

In another preferred embodiment, a PAM sequence (adjacent "NGG" sequence of the Cas9/sgRNA complex recognizing the target DNA site, N representing any base; preferably AGG) is also included in the formula I.

In another preferred embodiment, the nucleic acid construct has the structure of formula I':

S-PAM-M-S-PAM, formula I'.

In another preferred embodiment, the nucleic acid construct has the structure of formula II':

S-PAM-5' HA-M-3' HA-S-PAM, formula II '.

In another preferred example, the RNA marker sequence is MS2 sequence unit, PP7 sequence unit, boxB sequence unit and U1A sequence unit.

In another preferred embodiment, the RNA marker sequence comprises several stem-loop repeats, binding multiple copies of the RNA binding protein, thereby enriching each RNA molecule for a sufficiently strong fluorescent signal for visual detection. Comprises at least 6 RNA stem-loop repetitive sequences; preferably, at least 12 stem-loop repeats are included; more preferably, at least 17 stem-loop repeats are included.

In another preferred example, the MS2 sequence unit comprises at least 6 MS2 repeat sequences; preferably, at least 12 MS2 repeats are included; more preferably, at least 17 MS2 repeats are included.

In another preferred embodiment, the PP7 sequence unit comprises at least 6 PP7 repeats; preferably, at least 12 PP7 repeats are included; more preferably, at least 17 PP7 repeats are included.

In another preferred example, the boxB sequence unit comprises at least 6 boxB repeated sequences; preferably, at least 12 boxB repeats are included; more preferably, at least 17 boxB repeats are included.

In a third aspect of the invention, there is provided an expression cassette comprising a nucleic acid construct according to the second aspect of the invention.

Preferably, the expression cassette is cleavable by Cas9 in the cell to release the fragmented M sequence as a recombinant donor template. During knock-in, the M sequence is inserted into the rDNA region by either homology arm-mediated DNA repair (HDR) or non-homology arm-mediated (HITI) DNA repair.

In a fourth aspect of the invention, there is provided an expression vector comprising an expression cassette as described in the third aspect of the invention or a nucleic acid construct as described in the second aspect of the invention.

In another preferred embodiment, the expression vector is a plasmid, or a viral vector.

In another preferred embodiment, the viral vector is a lentiviral vector, an adenoviral vector, a herpesvirus vector, a poxviral vector, or an adeno-associated viral vector.

In a fifth aspect, the invention provides a kit for preparing a genetically engineered cell, the kit comprising an expression vector according to the fourth aspect of the invention, an expression cassette according to the third aspect of the invention, or a nucleic acid construct according to the second aspect of the invention.

In another preferred example, the kit further comprises a Cas9 protein expression plasmid.

In another preferred example, the kit further includes a first sgRNA expression plasmid that targets the S element of the nucleic acid construct.

In another preferred embodiment, the first sgRNA sequence is shown in SEQ ID No. 11.

In another preferred example, the gene encoding the Cas9 protein and the gene encoding the first sgRNA are integrated on the same plasmid.

In another preferred example, the kit further includes a second sgRNA expression plasmid that targets rDNA; preferably, the rDNA is eukaryotic; preferably mammalian (e.g., human) rDNA.

In another preferred example, the gene encoding the Cas9 protein, the gene encoding the first sgRNA, and the gene encoding the second sgRNA are integrated on the same plasmid.

Preferably, the second sgRNA targets rDNA at a site that is: 5 ″ -ETS region, 18S region, ITS1 region, 5.8S region, ITS2 region, 28S region, or 3' ETS region; preferably is located 5'ETS region, ITS1 region, 5.8S region, or 3' ETS region; more preferably in the 5.8S region.

In a sixth aspect, the invention provides a use of the genetically engineered cell of the first aspect of the invention in screening drugs.

In another preferred embodiment, the agent has the ability to inhibit ribosomal RNA production.

In another preferred embodiment, the drug has the ability to inhibit ribosome production.

In another preferred embodiment, the drug has the potential to inhibit cell proliferative activity.

In another preferred embodiment, the drug is an anti-tumor drug.

In another preferred embodiment, the agent has the ability to inhibit cleavage of ribosomal RNA precursors.

In another preferred embodiment, the agent has the ability to promote rDNA transcription.

The seventh aspect of the present invention provides a method for screening a compound based on live cell imaging, the method comprising the steps of:

(i) Contacting a compound to be screened with the genetically engineered cell according to the first aspect of the invention, and

(ii) And observing the fluorescence intensity indicating the rRNA synthesis level in the nucleus of the genetically engineered cell.

If the fluorescence intensity is reduced, the compound has potential activity of inhibiting cell proliferation, and if the fluorescence intensity is increased, the compound has potential activity of inhibiting rRNA shearing or promoting rRNA transcription.

In another preferred embodiment, the cell proliferation-inhibiting activity comprises an anti-tumor activity, an anti-inflammatory activity.

The eighth aspect of the present invention provides a method for preparing a genetically engineered cell, the method comprising the steps of:

(i) Providing a first genetically engineered cell capable of stably expressing the fusion protein, and

(ii) Inserting exogenous donor DNA capable of transcribing an RNA marker sequence into the rDNA of the first genetically engineered cell;

wherein the fusion protein is an RNA binding protein-fluorescent protein fusion protein;

the exogenous donor DNA can express an RNA marker sequence along with rDNA transcription; and, the RNA binding protein specifically recognizes the RNA marker sequence. Thereby realizing rRNA visualization.

In another preferred example, the fluorescent protein is a red fluorescent protein (e.g., tdTomato, dsRed, mCherry), a green fluorescent protein (e.g., eGFP, zsGreen), a Yellow Fluorescent Protein (YFP), a Blue Fluorescent Protein (BFP), a cyan fluorescent protein gene (CFP), etc.

In another preferred example, the RNA marker sequence is MS2 sequence unit, PP7 sequence unit, and boxB sequence unit.

In another preferred embodiment, the insertion site of the foreign coding gene in the rDNA is selected from the group consisting of: 5 ″ -ETS region, 18S region, ITS1 region, 5.8S region, ITS2 region, 28S region, or 3' ETS region; preferably 5'ETS region, ITS1 region, 5.8S region, or 3' ETS region; more preferably in the 5.8S region.

In another preferred embodiment, the RNA binding protein is an MCP protein.

In another preferred example, in step (ii), a CRISPR/Cas tool is used to insert foreign DNA into rDNA of the first genetically engineered cell.

In another preferred example, the CRISPR/Cas tool is a CRISPR/Cas9 gene editing tool.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

The following drawings are included to illustrate specific embodiments of the invention and are not intended to limit the scope of the invention as defined by the claims.

FIG. 1 is a schematic representation of ribosomal RNA visualization system design. NORs (nucleolar tissue regions) are the origin of the nucleoli, which consists of discrete repeats of rDNA clusters. The system can realize the specificity visualization of rRNA in living cells. And integrating the MS2 sequence into different regions of rDNA by using a CRISPR knock-in technology, transcribing the rDNA into rRNA, and combining the rRNA with stdMCP-tdTomato fusion protein to realize the visualization of the rRNA.

FIG. 2 is a technical route for implementation of the ribosomal RNA visualization system.

(a) Schematic representation of a donor plasmid for HITI repair mode;

(b) Obtaining virus particles in HEK293T cells through lentivirus packaging, and then obtaining a stable cell line 1 capable of expressing stdMCP-tdTomato fusion protein through lentivirus infection and screening of single cell clone;

(c) Constructing an rRNA visualization system in a stable cell line 1 by transient transfection and microscopic imaging of expression plasmids such as sgRNA, spCas9 and donor plasmids;

(d) Transient transfection and monoclonal screening of expression plasmids such as sgRNA, spCas9 and donor plasmids and the like to obtain a stable cell line 2,3 and 4 capable of stably marking rRNA;

(e) After the marking of rRNA in living cells is realized, rRNA becomes less and even disappears after the anti-tumor small molecule medicine is added.

Fig. 3 shows the optimization process for CRISPR tap-in technique.

When the rDNA region is edited by the CRISPR knock-in technique, nucleolus damage occurs, and the occurrence ratio of the nucleolus damage is expected to be reduced, so that the CRISPR knock-in technique is optimized.

(a) Co-localization of rRNA (red) with nucleolar component RPA43 (representing the fiber center), B23 (representing the granule component) and nucleolar morphology at donor plasmid and sgRNA concentrations of 5ng/10ng/20ng/50ng/100 ng;

(b) Change in cumulative intensity of fluorescently labeled 3' ETS rRNA when the concentration of donor plasmid and sgRNA is 5ng/10ng/20ng/50ng/100 ng;

(c) Change in cumulative intensity of fluorescently-labeled 5.8S rRNA when the concentration of donor plasmid and sgRNA is 5ng/10ng/20ng/50ng/100 ng;

(d) Labelling efficiency of 3' ETS rRNA when the donor plasmid and sgRNA concentrations are 5ng/10ng/20ng/50ng/100 ng;

(e) Labeling efficiency of 5.8S rRNA when the donor plasmid and sgRNA concentrations were 5ng/10ng/20ng/50ng/100 ng;

(f) Nucleolar damage rate of 3' ETS rRNA labelled cells when donor plasmid and sgRNA concentrations were 5ng/10ng/20ng/50ng/100 ng;

(g) Nucleolar damage rate of 3' ETS rRNA-labeled cells when the concentration of donor plasmid and sgRNA was 5ng/10ng/20ng/50ng/100 ng;

FIG. 4 is a depiction of co-localization of rRNA of three rRNA stably labeled cells with three nucleolar components.

(a) Co-localization of three rRNAs with three components of nucleolus, RPA43 (representing fiber center), FBL (representing dense fiber component), B23 representing granule component);

(b) For co-localization analysis of rRNA (red) and B23 (blue) in (a).

FIG. 5 is a graph showing the confirmation that stdMCP-tdTomato indeed labels nascent rRNA by treatment of rRNA stably labeled cells with Pol I inhibitors.

(a) A representative image of the change of the fluorescence intensity of a newborn rRNA (red) signal with time after adding Pol I transcription inhibitor Actinomycin D into ITS1rRNA stable labeled cells;

(b) Adding Pol I transcription inhibitor Actinomycin D into ITS1rRNA stable labeled cells to obtain a statistical image of the change of the fluorescence intensity of newborn rRNA (red) signals along with time;

(c) Statistical image of the change in fluorescence intensity of the nascent rRNA (red) signal with time after addition of the Pol I transcription inhibitor Actinomycin D to 3' ETS, ITS1,5.8S rRNA stably labeled cells.

FIG. 6 is an image of the change in fluorescence intensity of neonatal rRNA (red) signal with time and statistics of the change in red fluorescence intensity after visualization of rRNA in different cell lines followed by addition of the Pol I transcription inhibitor, actinomycin D (analysis of the change in fluorescence intensity of single and multiple cells with time).

(a) Representative image of fluorescence intensity of ETS nascent rRNA (red) signal 5' after addition of Pol I transcription inhibitor Actinomycin D as a function of time in U2OS cell line;

(b) Statistical images of fluorescence intensity of ETS nascent rRNA (red) signal 5' after addition of Pol I transcription inhibitor Actinomycin D to U2OS cell line over time;

(c) Representative image of fluorescence intensity of ETS nascent rRNA (red) signal 5' after addition of Pol I transcription inhibitor Actinomycin D as a function of time in REP-1 cell line;

(d) Statistical images of fluorescence intensity of ETS nascent rRNA (red) signal 5' after addition of Pol I transcription inhibitor Actinomycin D to REP-1 cell line over time.

FIG. 7 is a graph demonstrating the feasibility of stably-tagged rRNA single cell clones indicating high and low rDNA transcript levels.

3' -ETS, ITS1rRNA stably labeled cells the percentage of cells containing rRNA (red) signal after gene expression and the change in rRNA (red) fluorescence intensity over the whole cells were reduced with shRNA.

(a) Representative images of changes in rRNA (red) after reducing gene expression with shRNA for 3' -ETS rRNA stable labeled cells.

(b) Representative images of changes in rRNA (red) after shRNA reduced gene expression for ITS1rRNA stable marker cells.

(c) Statistical images of the percent change of cells containing rRNA (red) signal after reducing gene expression with shRNA for 3' -ETS rRNA stable labeled cells.

(d) A statistical image of the change in fluorescence intensity of rRNA (red) signals after gene expression is reduced with shRNA for 3' -ETS rRNA stably labeled cells.

(e) Statistical images of the percent change in cells containing rRNA (red) signal after reduction of gene expression with shRNA for ITS1rRNA stable labeled cells.

(f) A statistical image of rRNA (red) signal fluorescence intensity changes after gene expression is reduced by shRNA for 3' -ETS rRNA stable labeled cells.

(g) Statistical images to verify the effect of shRNA on reducing gene expression using real-time fluorescent quantitative PCR (qPCR).

(h) To verify that shRNA reduces the statistical image of the amount of 45spre-rRNA expression in gene-expressing cells using real-time fluorescent quantitative PCR (qPCR).

FIG. 8 is an image of the change in rRNA (red) signal fluorescence intensity of three rRNA stably labeled cells after addition of various anti-tumor small molecule drugs and a statistical result of the change in red fluorescence intensity of the whole cells (analysis result of the change in fluorescence intensity of the whole cells).

(a) A representative image of rRNA (red) signal fluorescence intensity change after a plurality of anti-tumor small molecule drugs are added into three rRNA stable labeled cells;

(b) And (3) adding a plurality of anti-tumor small molecule drugs into the three rRNA stable labeled cells to obtain the statistical result of the change of the rRNA (red) signal fluorescence intensity.

FIG. 9 is a graph showing the change in fluorescence intensity of rRNA (red) signal added with the antitumor drug CX-5461 after visualizing rRNA transcribed from different regions of rDNA, and a statistical result of the change in fluorescence intensity of red in the whole cell (analysis result of the change in fluorescence intensity in the whole cell).

(a) The structural diagram of different regions of rDNA and the diagram of the change of rRNA (red) signal fluorescence intensity after the antitumor drug CX-5461 is added are shown.

(b) Is a structural schematic diagram of different regions of rDNA and a statistical result of the change of the whole red fluorescence intensity of the cells after the anti-tumor drug CX-5461 is added.

FIG. 10 is an image of changes in fluorescence intensity of rRNA (red) signals of three rRNA-stably labeled cells after addition of anti-tumor small molecule drug 5-Fluorooracil that inhibits cleavage of rRNA precursors, and a statistical result of the percentage of cells containing rRNA (red) signals and the change in red fluorescence intensity of the whole cells (analysis result of the change in fluorescence intensity of the whole cells).

(a) A representative image of rRNA (red) signal fluorescence intensity change after adding an anti-tumor small molecule drug 5-Fluorooracil for inhibiting rRNA precursor shearing into three rRNA stable marked cells;

(b) Statistics of percentage change of rRNA (red) signal-containing cells after addition of anti-tumor small molecule drug 5-Fluorooracil that inhibits cleavage of rRNA precursors to three rRNA-stably labeled cells.

(c) And (3) statistics of rRNA (red) signal fluorescence intensity changes after adding an anti-tumor small molecule drug 5-Fluorooracil for inhibiting rRNA precursor shearing into the three rRNA stable labeled cells.

Fig. 11 is an image of change in rRNA (red) signal fluorescence intensity of three rRNA stably labeled cells after starvation treatment with nutrient serum added and a statistical result of change in red fluorescence intensity of the whole cells (analysis result of change in fluorescence intensity of the whole cells).

(a) Representative images of changes in fluorescence intensity of rRNA (red) signals after starvation treatment of three rRNA-stabilized labeled cells with nutrient serum added;

(b) The statistical result of the change of the rRNA (red) signal fluorescence intensity after the nutrient substance serum is added for the starvation treatment of the three rRNA stable marker cells.

The significance analysis is also stated: ^* p < 0.05 shows that there is a statistical difference, ^** p is less than 0.01, which shows significant statistical difference, ^*** p < 0.001 is very statistically significant, and n.s. represents no significant difference.

Detailed Description

The present inventors have conducted extensive and intensive studies to establish a novel efficient screening system targeting Pol I transcription, i.e., rRNA synthesis. The screening system is based on a newly developed living cell rRNA fluorescent reporter molecule, the expression change of rRNA can be directly imaged in the screening process, and the inhibition effect of small molecule drugs can be more quickly and sensitively reflected. Compared with the traditional screening strategy, the system greatly simplifies the screening process and time and greatly improves the screening efficiency. And three different regions of rDNA are stably inserted through donor plasmids to obtain a stable cell line capable of stably marking rRNA, and the stable cell line is sensitive to small molecule drugs. The present invention has been completed based on this finding.

Ribosomal DNA

Ribosomal DNA (rDNA) is a DNA sequence encoding Ribosomal RNA (rRNA). Ribosomal RNA (rRNA), the most abundant RNA in cells, binds to proteins to form ribosomes, which function to synthesize amino acids into peptide chains under the direction of mRNA. Ribosomes are a combination of proteins and rRNA molecules, components that translate mRNA molecules to produce proteins. The rDNA of eukaryotes includes a unit segment, an operon, and tandem repeats consisting of 5'ETS,18S, ITS1,5.8S, ITS2, 28S, 3' ETS tracts.

RNA labeling

RNA labeling is to covalently link a label (such as a labeling sequence and fluorescein) to RNA, and then realize the purpose of RNA identification and detection by detecting the label. RNA markers are widely used in the field of molecular detection.

The ideal RNA labeling method should meet the following requirements: the operation is simple, and the sensitivity is high; does not affect the specificity of base pairing; does not affect the activity of RNA; has no influence or little influence on the activity of the enzymatic reaction; the detection method has high sensitivity and specificity; the marker is harmless to human body.

For labeling of RNA in living cells, imaging systems for RNA labeling sequences and RNA Binding Proteins (RBPs) are commonly used. RNA marker sequences typically include multiple distinct RNA Stem-loop structural (Stem-loop) units that are specifically recognized by specific RNA binding proteins.

There are many RNA stem-loop building blocks, such as MS2, PP7, boxB and U1A. In general, in order to label a target RNA, it is necessary to insert multiple tandem stem-loop structural units into a target gene, for example, an untranslated region (e.g., intron, 5'UTR, 3' UTR, etc.) of the target gene. By transcription, the transcribed RNA carries multiple RNA stem-loop building blocks. RNA binding proteins are capable of specifically recognizing these RNA stem-loop building blocks. For example, MCP proteins are capable of specifically binding to MS2 stem-loop building blocks; the PCP protein can be specifically combined with a PP7 stem-loop structural unit; the lambda-N protein is capable of specifically binding to the boxB stem-loop building block.

In a preferred embodiment, the RNA marker sequence is 17XMS2V5.

In a preferred embodiment, the 1XPP7 sequence is as follows:

GGAGCAGACGATATGGCGTCGCTCC(SEQ ID NO.15)。

in a preferred embodiment, the 1xbox b sequence is as follows:

GGGCCCTGAAGAAGGGCCC(SEQ ID NO.16)。

typically, one or more (e.g., 1 to 3) copies of a fluorescent protein are fused to an RNA-binding protein, and multiple copies of a target RNA are linked in tandem to allow formation of fluorescent spots within the cell.

Genetically engineered cell and preparation method thereof

In a preferred embodiment of the invention, the invention provides a genetically engineered cell having incorporated into its rDNA a foreign DNA sequence (e.g., MS 2) for effecting RNA labeling. In addition, the genetically engineered cell also expresses a fusion protein that specifically binds to the RNA marker sequence.

In a preferred embodiment, the fusion protein comprises an RNA binding protein and a fluorescent protein that specifically bind to an RNA marker sequence. The fluorescent protein can be red fluorescent protein (e.g., tdTomato, dsRed, mCherry), green fluorescent protein (e.g., eGFP, zsGreen), yellow Fluorescent Protein (YFP), blue Fluorescent Protein (BFP), cyan fluorescent protein gene (CFP), etc.

The genetically engineered cell of the present invention is prepared by Gene Knock-in (Gene Knock-in). Gene Knock-in (Gene Knock-in) is mainly to introduce exogenous genes into the genome of cells by means of random integration, a transposon system, homologous recombination and the like, thereby realizing the change of Gene functions.

The process of preparing the genetically engineered cell is preferably a site-directed knock-in of a gene using CRISPR/Cas system tools. The CRISPR/Cas system is an acquired immune system which is found in most bacteria and all archaea, and is named as a clustered regular interspersed short palindromic repeat/clustered protein system (CRISPR-associated proteins). A plurality of different types of CRISPR/Cas systems are found, wherein the CRISPR/Cas9 system is composed of a Cas9 protein and a guide RNA (gRNA) as a core, and only a single cleavage protein (Cas 9) is required for the cleavage of a foreign gene, so that the CRISPR/Cas system is most convenient to use. Binding of Cas9 protein to gRNA enables cleavage of DNA at specific sites, and the last 3 NGG sequence of its recognition site is called PAM (protospaceradjacentmotif) sequence, which is very important for DNA cleavage.

Genome editing is carried out by using a CRISPR/Cas9 system, a Cas9 protein with DNA (deoxyribonucleic acid) cutting enzyme activity is heterologously expressed in a cell, and then sgRNA and a target point homologous sequence are obtained to guide Cas9 to a target point for DNA cutting. Specific methods of operation are well known to those skilled in the art.

Specifically, sgRNA in the CRISPR/Cas9 system recognizes and binds to a target sequence of a target Gene, guides Cas9 to cut a binding site, generates a DNA double strand break (dsb), and introduces an exogenous donor DNA (donor sequence) into the target site of a genome in a fixed-point manner by an intracellular Gene recombination repair manner, thereby realizing Gene Knock-in (Gene Knock-in).

In a preferred embodiment of the present invention, the present invention provides a method for preparing the genetically engineered cell, the method comprising the steps of:

(i) Providing a first genetically engineered cell capable of stably expressing the fusion protein, and

(ii) Inserting exogenous DNA into rDNA of the first genetically engineered cell;

wherein the fusion protein is an RNA binding protein-fluorescent protein fusion protein;

the exogenous DNA can be transcribed along with rDNA to become an RNA marker sequence in rRNA; and, the RNA binding protein specifically recognizes the RNA marker sequence; in the step (ii), inserting an exogenous DNA into the rDNA of the first genetically engineered cell by using a CRISPR/Cas tool.

In a preferred embodiment of the invention, the invention provides a high-efficiency screening system which can be used for inhibiting cell proliferation, and the expression level of rRNA is visualized through fluorescent protein, so that the effect of targeting rRNA synthesis of small molecule drugs can be quantified in real time. Thereby identifying small molecules including, but not limited to, anti-tumor.

In a preferred embodiment, an exogenous RNA marker sequence (as a donor template, such as a 17xMS2V5 sequence (SEQ ID No. 1)) is inserted into the DNA sequence of a ribosomal RNA gene (also called rDNA) using CRISPR-Cas9 gene editing techniques.

In a preferred embodiment, the CRISPR-Cas9 based gene editing system inserts RNA marker sequences into specific regions within rDNA using either homology arm mediated DNA repair (HDR) or non-homology arm mediated (HITI) DNA repair.

In a more preferred embodiment, the repair template sequence (SEQ ID No. 2) is inserted into a specific region within rDNA using HDR means, e.g., targeting the ITS1 region.

In a more preferred embodiment, the repair template sequence (SEQ ID No. 3) is inserted into a specific region within rDNA using HITI format.

In a more preferred embodiment, the RNA marker sequence (17x MS2 sequence) is inserted into different rDNA regions, a specific sgRNA sequence is constructed, and a high efficiency sgRNA sequence is selected. According to the invention, efficient sgRNA sequences are respectively screened out aiming at different insertion sites, and each sgRNA sequence is shown as SEQID No. 4-SEQID No. 10.

In a preferred embodiment, the sgRNA is screened for the goal of identifying sgrnas that target rDNA with high efficiency. Sgrnas with high efficiency can be screened using the DNA labeling efficiency of the ribosomal rRNA gene (rDNA) as an index. Cells stably expressing dCas9-GFP14X were plated one day in eight well chamber cover slides (DMEM high glucose containing 10% fetal bovine serum and 1% double antibody). The next day, the cell density was about 40% to 60%, and the cells were cultured in DMEM without the double antibody and containing 10% fetal calf serum. Transfection was performed with PEI reagents, 400ng sgRNA plasmid was transfected. And on the third day, the rDNA labeling efficiency is observed, and sgRNA with high rDNA labeling efficiency is selected and used for labeling rRNA. For rRNA labeling, cells were plated on eight-well chamber cover slides and incubated overnight with 10% fetal bovine serum in DMEM and 1% double antibody. The cell density is about 40-60% on the next day, the culture medium is changed into DMEM without double antibody and containing 10% fetal bovine serum, fuGENE is used as transfection reagent, sgRNA corresponding to rDNA of different regions can be selected according to experiment requirements, and 2.5ng of Cas9,5ng/100ng of sgRNA,5ng/100ng of donor plasmid and 5ng/100ng of sgTS1 (donor plasmid is cut and linearized) are introduced into cells. After 48 hours of transient transfer, the cells were observed under a fluorescent microscope to determine whether the corresponding rRNAs were labeled. Adding an inhibitor Actinomycin D for inhibiting Pol I transcription, and judging whether the generation of rRNA can be well inhibited or not so as to verify that a signal point on an intracellular marker really represents the accumulation of rRNA.

In a preferred embodiment, the cell line is stably expressed with tdMCP-GFP (coding gene such as SEQ ID No. 12) or stdMCP-tdTomato (coding gene such as SEQ ID No. 13), so that rRNA molecules successfully inserted into the MS2 sequence can be combined by the tdMCP molecules, thereby realizing the rRNA living cell marking.

In order to obtain the best rRNA imaging system, single cell clones of tdMCP-GFP and stdMCP-tdTomato cell lines can be respectively further separated, and the single cell clone with the best rRNA imaging signal-to-noise ratio is obtained by screening.

rRNA is transcribed by RNA polymerase I (Pol I), and the screening system is mainly used for screening drugs taking Pol I as a target.

In a preferred embodiment, the steps for efficient screening of anti-tumor small molecule drugs are as follows:

1) After the cell group marked on the specific rRNA region is obtained, single cell clone is further established, and the single cell clone with good cell state and positive rRNA mark is screened. For example, a single cell clone stably tagged with 5.8S rRNA was created and confirmed by sequencing that the 17XMS2V5 sequence was indeed inserted into the designed site.

2) The 5.8S rRNA single cell clones were plated in 96-well plates and the next day the small molecules to be screened were added, one well for each small molecule. After the cells and the small molecules are incubated for three hours, the cells can be placed under a fluorescence microscope for screening, and whether rRNA signal points change or not is observed, so that the potential anti-tumor small molecules are identified.

In a preferred embodiment, the RNA marker sequence is the 17XMS2V5 sequence (SEQ ID No. 1) as follows:

GAAGGTGACAACCGGTAACCTACAAACGGGTGGAGGATCACCCCACCCGACACTTCACAATCAAGGGGTACAATACACAAGGGTGGAGGAACACCCCACCCTCCAGACACATTACACAGAAATCCAATCAAACAGAAGCACCATCAGGGCTTCTGCTACCAAATTTATCTCAAAAAACTACAACAAGGAATCACCATCAGGGATTCCCTGTGCAATATACGTCAAACGAGGGCCACGACGGGAGGACGATCACGCCTCCCGAATATCGGCATGTCTGGCTTTCGAATTCAGTGCGTGGAGCATCAGCCCACGCAGCCAATCAGAGTCGAATACAAGTCGACTTTCGCGAAGAGCATCAGCCTTCGCGCCATTCTTACACAAACCACACTCTCCCCTACAGGAACAGCATCAGCGTTCCTGCCCAGTACCCAACTCAAGAAAATTTATGTCCCCATGCAGCATCAGCGCATGGGCCCCAAGAATACATCCCCAACAAAATCACATCCGAGCACCAACAGGGCTCGGAGTGTTGTTTCTTGTCCAACTGGACAAACCCTCCATGGACCATCAGGCCATGGACTCTCACCAACAAGACAAAAACTACTCTTCTCGAAGCAGCATCAGCGCTTCGAAACACTCGAGCATACATTGTGCCTATTTCTTGGGTGGACGATCACGCCACCCATGCTCTCACGAATTTCAAAACACGGACAAGGACGAGCACCACCAGGGCTCGTCGTTCCACGTCCAATACGATTACTTACCTTTCGGGATCACGATCACGGATCCCGCAGCTACATCACTTCCACTCAGGACATTCAAGCATGCACGATCACGGCATGCTCCACAAGTCTCAACCACAGAAACTACCAAATGGGTTCAGCACCAGCGAACCCACTCCTACCTCAAACCTCTTCC。

in a preferred embodiment, the HDR repair template plasmid sequence for the 17xms2V5 sequence insertion is as follows (SEQ ID No. 2):

GCACCGATGCTCTCCGAGGAGGAATTCTTCGCTCGCTCGTTCGTTCGCCGCCCGGCCCCGCCGCCGCGAGAGCCGAGAACTCGGGAGGGAGACGGGGGGGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAAAGAAGGGCGTGTCGTTGGTGTGCGCGTGTCGTGGGGCCGGCGGGCGGCGGGGAGCGGTCCCCGGCCGCGGCCCCGACGACGTGGGTGTCGGCGGGCGCGGGGGCGGTTCTCGGCGGCGTCGCGGCGGGTCTGGGGGGGTCTCGGTGCCCTCCTCCCCGCCGGGGCCCGTCGTCCGGCCCCGCCGCGCCGGCTCCCCGTCTTCGGGGCCGGCCGGATTCCCGTCGCCTCCGCCGCGCCGCTCCGCGCCGCCGGGCACGGCCCCGCTCGCTCTCCCCGGCCTTCCCGCTAGGGCGTCTCGAGGGTCGGGGGCCGGACGCCGGTCCCCTCCCCCGCCTCCTCGTCCGCCCCCCCGCCGTCCAGGTACCTAGCGCGTTCCGGCGCGGAGGTTTAAAGACCCCTTGGGGGGATCGCCCGTCCGCCCGTGGGTCGGGGGCGGTGGTGGGCCCGCGGGGGAGTCCCGTCGGGAGGGGCCCGGCCCCTCCCGCGCCTCCACCGCGGACTCCGCTCCCCGGCCGGGGCCGCGCCGCCGCCGCCGCCGCGGCGGCCGTCGGGTGGGGGAAGCTTGAAGGTGACAACCGGTAACCTACAAACGGGTGGAGGATCACCCCAC CCGACACTTCACAATCAAGGGGTACAATACACAAGGGTGGAGGAACACCCCACCCTCCAGACACATTACACAGAAA TCCAATCAAACAGAAGCACCATCAGGGCTTCTGCTACCAAATTTATCTCAAAAAACTACAACAAGGAATCACCATC AGGGATTCCCTGTGCAATATACGTCAAACGAGGGCCACGACGGGAGGACGATCACGCCTCCCGAATATCGGCATGT CTGGCTTTCGAATTCAGTGCGTGGAGCATCAGCCCACGCAGCCAATCAGAGTCGAATACAAGTCGACTTTCGCGAA GAGCATCAGCCTTCGCGCCATTCTTACACAAACCACACTCTCCCCTACAGGAACAGCATCAGCGTTCCTGCCCAGT ACCCAACTCAAGAAAATTTATGTCCCCATGCAGCATCAGCGCATGGGCCCCAAGAATACATCCCCAACAAAATCAC ATCCGAGCACCAACAGGGCTCGGAGTGTTGTTTCTTGTCCAACTGGACAAACCCTCCATGGACCATCAGGCCATGG ACTCTCACCAACAAGACAAAAACTACTCTTCTCGAAGCAGCATCAGCGCTTCGAAACACTCGAGCATACATTGTGC CTATTTCTTGGGTGGACGATCACGCCACCCATGCTCTCACGAATTTCAAAACACGGACAAGGACGAGCACCACCAG GGCTCGTCGTTCCACGTCCAATACGATTACTTACCTTTCGGGATCACGATCACGGATCCCGCAGCTACATCACTTC CACTCAGGACATTCAAGCATGCACGATCACGGCATGCTCCACAAGTCTCAACCACAGAAACTACCAAATGGGTTCA GCACCAGCGAACCCACTCCTACCTCAAACCTCTTCCTCTAGACTTTACCCGGCGGCCGTCGCGCGCCTGCCGCGCGTGTGGCGTGCGCCCCGCGCCGTGGGGGCGGGAACCCCCGGGCGCCTGTGGGGTGGTGTCCGCGCTCGCCCCCGCGTGGGCGGCGCGCGCCTCCCCGTGGTGTGAAACCTTCCGACCCCTCTCCGGAGTCCGGTCCCGTTTGCTGTCTCGTCTGGCCGGCCTGAGGCAACCCCCTCTCCTCTTGGGCGGGGGGGGCGGGGGGACGTGCCGCGCCAGGAAGGGCCTCCTCCCGGTGCGTCGTCGGGAGCGCCCTCGCCAAATCGACCTCGTACGACTCTTAGCGGTGGATCACTCGGCTCGTGCGTCGATGAAGAACGCAGCTAGCTGCGAGAATTAATGTGATATCGCACCGATGCTCTCCGAGGAGG。

in a preferred embodiment, the HITI repair template plasmid sequence for 17XMS2V5 sequence insertion is as follows (SEQ ID No. 3):

GCACCGATGCTCTCCGAGGAGGGAAGGTGACAACCGGTAACCTACAAACGGGTGGAGGATCACCCCAC CCGACACTTCACAATCAAGGGGTACAATACACAAGGGTGGAGGAACACCCCACCCTCCAGACACATTACACAGAAA TCCAATCAAACAGAAGCACCATCAGGGCTTCTGCTACCAAATTTATCTCAAAAAACTACAACAAGGAATCACCATC AGGGATTCCCTGTGCAATATACGTCAAACGAGGGCCACGACGGGAGGACGATCACGCCTCCCGAATATCGGCATGT CTGGCTTTCGAATTCAGTGCGTGGAGCATCAGCCCACGCAGCCAATCAGAGTCGAATACAAGTCGACTTTCGCGAA GAGCATCAGCCTTCGCGCCATTCTTACACAAACCACACTCTCCCCTACAGGAACAGCATCAGCGTTCCTGCCCAGT ACCCAACTCAAGAAAATTTATGTCCCCATGCAGCATCAGCGCATGGGCCCCAAGAATACATCCCCAACAAAATCAC ATCCGAGCACCAACAGGGCTCGGAGTGTTGTTTCTTGTCCAACTGGACAAACCCTCCATGGACCATCAGGCCATGG ACTCTCACCAACAAGACAAAAACTACTCTTCTCGAAGCAGCATCAGCGCTTCGAAACACTCGAGCATACATTGTGC CTATTTCTTGGGTGGACGATCACGCCACCCATGCTCTCACGAATTTCAAAACACGGACAAGGACGAGCACCACCAG GGCTCGTCGTTCCACGTCCAATACGATTACTTACCTTTCGGGATCACGATCACGGATCCCGCAGCTACATCACTTC CACTCAGGACATTCAAGCATGCACGATCACGGCATGCTCCACAAGTCTCAACCACAGAAACTACCAAATGGGTTCA GCACCAGCGAACCCACTCCTACCTCAAACCTCTTCCGCACCGATGCTCTCCGAGGAGG。

in a preferred embodiment, the CRISPR sgRNA sequence for targeting a 5' ets insertion is as follows (SEQ ID No. 4):

ACACGCACGGCACGGAGCCAGC。

in a preferred embodiment, the CRISPR sgRNA sequence for targeted ITS1 insertion is as follows (SEQ ID No. 5):

CGGGTGGGGGCTTTACCCGG。

in a preferred embodiment, the CRISPR sgRNA sequence for targeted ITS2 insertion is as follows (SEQ ID No. 6):

CGTCCCGAGCTTCCGCGTCG。

in a preferred embodiment, the CRISPR sgRNA sequence for targeting the 18S insertion is as follows (SEQ ID No. 7):

CGACCCGGGGAGGTAGTGA。

in a preferred embodiment, the CRISPR sgRNA sequence for targeting a 5.8S insertion is as follows (SEQ ID No. 8):

CGACACTTCGAACGCACTTG。

in a preferred embodiment, the CRISPR sgRNA sequence for targeted 28S insertion is as follows (SEQ ID No. 9):

CGGCCGAGGTGGGATCCCG。

in a preferred embodiment, the CRISPR sgRNA sequence for targeting 3' ets insertion is as follows (SEQ ID No. 10):

GGAGCGTGGTTTGGGAGCCG。

in a preferred embodiment, the CRISPR sgRNA sequence used for targeted repair template plasmid linearization is as follows (SEQ ID No. 11):

GCACCGATGCTCTCCGAGG。

in a preferred embodiment, the gene sequence of the tdMCP-GFP fusion protein is as follows (SEQ ID No. 12):

ATGGCTTCTAACTTTACTCAGTTCGTTCTCGTCGACAATGGCGGAACTGGCGACGTGACTGTCGCCCCAAGCAACTTCGCTAACGGGATCGCTGAATGGATCAGCTCTAACTCGCGTTCACAGGCTTACAAAGTAACCTGTAGCGTTCGTCAGAGCTCTGCGCAGAATCGCAAATACACCATCAAAGTCGAGGTGCCTAAAGGCGCCTGGCGTTCGTACTTAAATATGGAACTAACCATTCCAATTTTCGCCACGAATTCCGACTGCGAGCTTATTGTTAAGGCAATGCAAGGTCTCCTAAAAGATGGAAACCCGATTCCCTCAGCAATCGCAGCAAACTCCGGCATCTACGCCATGGCTTCTAACTTTACTCAGTTCGTTCTCGTCGACAATGGCGGAACTGGCGACGTGACTGTCGCCCCAAGCAACTTCGCTAACGGGATCGCTGAATGGATCAGCTCTAACTCGCGTTCACAGGCTTACAAAGTAACCTGTAGCGTTCGTCAGAGCTCTGCGCAGAATCGCAAATACACCATCAAAGTCGAGGTGCCTAAAGGCGCCTGGCGTTCGTACTTAAATATGGAACTAACCATTCCAATTTTCGCCACGAATTCCGACTGCGAGCTTATTGTTAAGGCAATGCAAGGTCTCCTAAAAGATGGAAACCCGATTCCCTCAGCAATCGCAGCAAACTCCGGCATCTACGCGGATTCTAGAATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA。

in a preferred embodiment, the gene sequence of stdMCP-tdTomato fusion protein is as follows (SEQ ID No. 13):

ATGGGCCCAAAAAAGAAAAGAAAAGTTGGCTACCCCTACGACGTGCCCGACTACGCCATCGAAGGCCGCCATATGCTAGCCGCTTCTAACTTTACTCAGTTCGTTCTCGTCGACAATGGCGGAACTGGCGACGTGACTGTCGCCCCAAGCAACTTCGCTAACGGGATCGCTGAATGGATCAGCTCTAACTCGCGTTCACAGGCTTACAAAGTAACCTGTAGCGTTCGTCAGAGCTCTGCGCAGAATCGCAAATACACCATCAAAGTCGAGGTGCCTAAAGGCGCCTGGCGTTCGTACTTAAATATGGAACTAACCATTCCAATTTTCGCCACGAATTCCGACTGCGAGCTTATTGTTAAGGCAATGCAAGGTCTCCTAAAAGATGGAAACCCGATTCCCTCAGCAATCGCAGCAAACTCCGGCATCTACGCCATGGCCAGCAACTTCACCCAGTTCGTGCTGGTGGACAACGGCGGCACCGGCGACGTGACCGTGGCCCCCAGCAACTTCGCCAACGGCATCGCCGAGTGGATCAGCAGCAACAGCAGAAGCCAGGCCTACAAGGTGACCTGCAGCGTGAGACAGAGCAGCGCCCAGAACAGAAAGTACACCATCAAGGTGGAGGTGCCCAAGGGCGCCTGGAGAAGCTACCTGAACATGGAGCTGACCATCCCCATCTTCGCCACCAACAGCGACTGCGAGCTGATCGTGAAGGCCATGCAGGGCCTGCTGAAGGACGGCAACCCCATCCCCAGCGCCATCGCCGCCAACAGCGATTCTAGAATGGTGAGCAAGGGCGAGGAGGTCATCAAAGAGTTCATGCGCTTCAAGGTGCGCATGGAGGGCTCCATGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCCCAGTTCATGTACGGCTCCAAGGCGTACGTGAAGCACCCCGCCGACATCCCCGATTACAAGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGTCTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCACGCTGATCTACAAGGTGAAGATGCGCGGCACCAACTTCCCCCCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCACCGAGCGCCTGTACCCCCGCGACGGCGTGCTGAAGGGCGAGATCCACCAGGCCCTGAAGCTGAAGGACGGCGGCCACTACCTGGTGGAGTTCAAGACCATCTACATGGCCAAGAAGCCCGTGCAACTGCCCGGCTACTACTACGTGGACACCAAGCTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAGCGCTCCGAGGGCCGCCACCACCTGTTCCTGGGGCATGGCACCGGCAGCACCGGCAGCGGCAGCTCCGGCACCGCCTCCTCCGAGGACAACAACATGGCCGTCATCAAAGAGTTCATGCGCTTCAAGGTGCGCATGGAGGGCTCCATGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCCCAGTTCATGTACGGCTCCAAGGCGTACGTGAAGCACCCCGCCGACATCCCCGATTACAAGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGTCTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCACGCTGATCTACAAGGTGAAGATGCGCGGCACCAACTTCCCCCCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCACCGAGCGCCTGTACCCCCGCGACGGCGTGCTGAAGGGCGAGATCCACCAGGCCCTGAAGCTGAAGGACGGCGGCCACTACCTGGTGGAGTTCAAGACCATCTACATGGCCAAGAAGCCCGTGCAACTGCCCGGCTACTACTACGTGGACACCAAGCTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAGCGCTCCGAGGGCCGCCACCACCTGTTCCTGTACGGCATGGACGAGCTGTACAAGtaa。

applications of the invention

The genetic engineering cell provided by the invention has foreign DNA capable of forming an RNA marker sequence through transcription integrated in rDNA, and also expresses fusion protein specifically combined with the RNA marker sequence. The fusion protein comprises RNA binding protein and fluorescent protein which are specifically combined with RNA marker sequence.

The expression of ribosomal RNA can be directly observed at the single cell level by culturing the genetically engineered cell of the invention and using a fluorescence imaging method. When Pol I transcription inhibitors are added to the genetically engineered cell culture fluid, the fluorescence level is significantly reduced or even eliminated. Therefore, the genetically engineered cell of the invention can directly observe the effect of the small molecule drug on the expression of the ribosomal RNA at the single cell level, thereby being used for screening the drug.

The main advantages of the invention are:

(1) The CRISPR-Cas9 editing technology is adopted for the first time, the visualization of rRNA of living cells is realized, and a simple and efficient rDNA-targeted CRISPR-Cas9 editing system is established.

(2) A fluorescence reporting system of living cell rRNA is established, and the expression level of rRNA can be quantified in real time by a fluorescence imaging method.

(3) A small molecule drug screening system based on living cell rRNA fluorescent reporter molecules is created. The system directly observes the effect of the micromolecule drug on the ribosomal RNA expression at the level of single cell by using a fluorescence imaging method without detecting by real-time fluorescence quantitative PCR or Northern blot hybridization, thereby improving the sensitivity of screening and shortening the time for primarily screening the drug;

(4) Three rRNA-labeled stable cell lines are established, wherein the 5.8S rRNA-labeled stable cell line has stronger rRNA signals, fluorescence is not easy to quench due to phototoxicity, and the method is very suitable for small molecule screening under fluorescence imaging. The stable cell line is sensitive to a variety of known anti-tumor small molecule drugs. The establishment of the rRNA marked stable cell line greatly simplifies the screening process and greatly improves the screening efficiency.

(5) The screening system has definite target, the screening thought is based on a sufficient theoretical basis, the screening system is favorable for discovering small molecules influencing rRNA generation or rRNA shearing, the small molecules have the potential of inhibiting or promoting cell proliferation, differentiation and the like, and the small molecules obtained by screening include but are not limited to the discovery of anti-tumor small molecules.

The present invention will be described in further detail with reference to the following examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Parts and percentages are by weight unless otherwise indicated.

Example 1:

(1) Construction of a donor plasmid with non-homologous arms mediating DNA repair, as shown in fig. 2 (a):

the method is characterized in that 17 copies of MS2V5 tandem arrangement sequences (donor sequences) are assembled into a common plasmid vector by a molecular cloning construction means, a screened DNA sequence (GCACCGATGCTCCGAGGAGGG, SEQ ID No. 14) which is 22 bases and can be efficiently sheared by Cas9 is added at both ends of an MS2 fragment by a PCR amplification method, and the sequence comprises a target sequence and a PAM locus of the Cas 9. Meanwhile, an sgRNA (GCACCGATGCTCCGAGG, SEQ ID NO. 11) capable of identifying the sequence is constructed, which is referred to as sgTS1 for short. The donor plasmid and the Cas9/sgTS1 are co-expressed in cells, the donor plasmid can be cut by the Cas9, so that the DNA of a donor template is fragmented, and the efficiency of inserting MS2 into rDNA in a non-homologous recombination mode is greatly improved.

(2) Construction of donor plasmids for homology arm mediated DNA repair:

the following three fragments were cloned by homologous recombination molecular cloning: 1) 17 copies of MS2V5; 2) A homologous arm with the length of 300-800bp at the 5' end of the rDNA insertion site; 3) Homologous arms with the length of 300-800bp at the 3' end of the rDNA insertion site are spliced into a fragment, and a universal vector is inserted. A screened DNA sequence (GCACCGATGCTCCGGAGGAGG, SEQ ID NO. 14) with the length of 22 bases and capable of being efficiently sheared by Cas9 is respectively added at two ends of the homologous arm through a PCR amplification method, and the sequence comprises a target sequence and a PAM locus of the Cas 9. The donor plasmid and the Cas9/sgTS1 are co-expressed in cells, the donor plasmid can be cut by the Cas9, so that the DNA of a donor template is fragmented, and the efficiency of inserting MS2 into rDNA in a homologous recombination mode is greatly improved.

Example 2:

(1) Constructing a visual stable cell line as shown in fig. 2 (b):

(1.1) on the day, culturing HEK293T cells (purchased from ATCC) by using a streptomycin-free mixed solution (PS) culture medium and paving the HEK293T cells into a 12-well plate to form a first cell culture medium (the HEK293T cells are added at a concentration which can enable the cell density to reach more than 80% on the day);

(1.2) the following day, lentivirus packaging was performed: premixing 750ng of stdMCP-tdTomato virus expression plasmid (construction method reference PMID: 33104788), 705ng of pCMV-dR8.91 plasmid (purchased from Addgene) and 87ng of PMD2.G plasmid (purchased from Addgene) by using 75ul of serum-reduced medium (Opti-medium), adding 4.5ul of Fugene transient transfection reagent (Promega) to form liposome, dropwise adding the liposome into the supernatant of the first cell culture medium, and performing transient transfection on HEK293T cells in the first cell culture medium (preparing cells containing virus particles);

(1.3) after 12 hours of transient transfection in step (1.2), aspirating the first cell culture medium supernatant and replacing the first cell culture medium with fresh medium;

(1.4) culturing the cells to be infected after 48 hours of transient transfection in the step (1.2) and paving the cells to be infected into a 24-well plate to form a second cell culture medium (the concentration of the cells to be infected is added to enable the next day cell density to reach about 60%);

(1.5) on the day after 24 hours of forming the second cell culture medium, sucking the first cell culture medium supernatant obtained by replacing the fresh culture medium in the step (1.3), then placing the first cell culture medium supernatant into a centrifuge for 8 minutes at the rotating speed of 800g, taking the supernatant after centrifugation, and extracting the cell supernatant containing the virus;

(1.6) preparing a PS-free culture medium containing a gene transfection enhancer (polybrene) with a concentration of 5 [ mu ] g/mL as a third culture medium to replace the second cell culture medium, so that a cell to be infected, which uses human HeLa cells (purchased from ATCC), is cultured and transferred to the third culture medium, and then 30-90 ul of cell supernatant containing the virus is taken and added dropwise to the third culture medium supernatant infected with the cell to be infected (to obtain infected cells);

(1.7) after 12 hours of infection in the step (1.6), replacing a fresh culture medium for the third culture medium (of the infected cells) and continuing culturing until the cells can be passaged into an 8-well plate for microscopic imaging observation, screening a cell infection population according to fluorescence intensity and infection efficiency, and separating single-cell clones of a 96-well plate;

the screening of the single cell clone specifically screens out cell infected groups with proper fluorescence intensity and higher infection efficiency: and selecting the cells in the single hole of the pore plate with the preserved infection efficiency of more than 30-40%, otherwise, removing.

(1.8) when the cells in the single well of the 96-well plate grow to the state of cell groups, observing the fluorescence intensity under a microscope, and screening the single-cell clone.

The screening standard of the single cell clone refers to RNA marking signal to noise ratio, stdMCP-tdTomato single cell clone with the best signal to noise ratio is used for rRNA marking, and the single cell clone is a cell line 1.

(2) Transient visualization of rRNA in stable cell line 1, as shown in fig. 2 (c):

(2.1) rRNA cells to be marked are cultured by using a streptomycin-free mixed solution (PS) culture medium and are paved into an eight-hole chamber cover glass slide to form a fourth cell culture medium (the adding concentration of the cells can enable the cell density to reach more than 40% on the next day);

(2.2) on the day 24 hours after the start of the culture in step (2.1), premixing 100ng each of sgRNA expression plasmid (for targeting the target endogenous gene and the donor plasmid), 2.5ng SpCas9 protein expression plasmid (purchased from addge) and 100ng of the donor plasmid with 25ul of serum-reduced medium (Opti-medium), adding 1.5 μ l of Fugene transient transfection reagent (Promega) to form liposomes, slowly adding dropwise the liposomes to the supernatant of the fourth cell culture medium, and transiently transfecting the rRNA cells to be labeled;

(2.3) after 48 hours from the completion of step (2.2), the cells obtained in step (2.2) were placed in a live microscope cell station set to a temperature of 37 ℃ and containing a volume fraction of 5% CO ₂ 。

The microscope also comprises a microscope light source and an imaging camera, and live cell imaging visualization is carried out by setting and adjusting the laser intensity of the microscope light source and the exposure time of the imaging camera.

rRNA appeared red, i.e., in the live cell state, rRNA was observed.

The rRNA cell to be labeled in this example refers to the stable cell line 1 of the visual system.

The donor plasmid is shown in a specific schematic diagram in (a) in FIG. 2: two ends are sgTS1 recognition sequence and PAM sequence thereof, and the middle is MS2V5 _17x And (4) sequencing. The result after preparation is shown in SEQ ID No.3.

Example 3

(1) Experimental conditions testing for visualization of 3' ETS rRNA in stable cell line 1, as shown in FIG. 2 (c):

(1.1) as in (2.1) - (2.2) steps in example 2, a donor plasmid was inserted into the 3'ets of the 47S rRNA precursor and expressed, wherein sgrnas to be used were sgrnas targeting the 3' ets region, such as SEQ ID No.10 and sgRNA-sgTS 1 targeting the donor plasmid (shown in SEQ ID No. 11), respectively, and the concentration of Cas9 was 2.5ng, but the concentrations of the donor plasmid and the sgRNA plasmid were five concentrations of 5ng,10ng,20ng,50ng,100ng, and 5 visual rRNA cells, such as cell 1, cell 2, cell 3, cell 4, and cell 5, respectively, were prepared.

(2) Different donor plasmids and sgRNA plasmid concentrations were labeled 3' ets rRNA and live cell imaging was performed:

(2.1) on the day 48 hours after completion of step (1.1), the fine powder obtained in step (1.1) was collectedCells 1,2,3,4,5 were placed in a confocal microscope live cell workstation set to a temperature of 37 ℃ and containing 5% by volume of CO ₂ .561 laser intensity is set to 5% and exposure time is 100ms.

The ratio of rRNA-labeled cells to total cells at different Cas9 concentrations was counted, and the results are shown in fig. 3 (d). The ratio of nucleolar damage in rRNA-labeled cells was counted at different concentrations of donor plasmid and sgRNA plasmid, and the results are shown in (f) of fig. 3. The change in the overall red fluorescence intensity of 3' ETS rRNA cells was counted for different concentrations of donor plasmid and sgRNA plasmid, and the results are shown in FIG. 3 (b).

The results show that when the concentrations of Cas9, sgRNA and donor plasmid are kept low, the labeling efficiency of rRNA and the overall red fluorescence intensity are affected, but the kernel damage rate can be reduced.

(3) Experimental condition testing for visualization of 5.8S rRNA in stable cell line 1, as shown in fig. 2 (c):

(3.1) as in (2.1) - (2.2) in example 2, 5.8S of the 47S rRNA precursor was inserted into a donor plasmid and expressed, wherein sgrnas to be used were sgrnas targeting the 5.8S region, such as SEQ ID No.8 and sgRNA-sgTS 1 targeting the donor plasmid (shown in SEQ ID No. 11), respectively, and the Cas9 plasmid concentration was 2.5ng, but the concentrations of the donor plasmid and the sgRNA plasmid were five concentrations of 5ng,10ng,2 ng,50ng,100ng, and 5 visual rRNA cells, such as cell 6, cell 7, cell 8, cell 9, and cell 10, respectively, were prepared.

(4) Different donor and sgRNA plasmid concentrations labeled 5.8S rRNA and live cell imaging:

(4.1) on the day 48 hours after completion of step (3.1), the cells 6,7,8,9, 10 obtained in step (3.1) were placed in a confocal microscope live cell workstation set to a temperature of 37 ℃ and containing a volume fraction of 5% CO ₂ .561 laser intensity is set to 5% and exposure time is 100ms.

The ratio of rRNA-labeled cells to the total cells was counted at different concentrations of donor plasmid and sgRNA plasmid, and the results are shown in (e) of FIG. 3. The proportion of nucleolar lesions in rRNA-labeled cells was counted at different concentrations of donor plasmid and sgRNA plasmid, and the results are shown in fig. 3 (g). The change in the overall red fluorescence intensity of 5.8S rRNA cells at different concentrations of donor plasmid and sgRNA plasmid was counted, and the results are shown in fig. 3 (c).

The results show that when the concentrations of Cas9, sgRNA and donor plasmid are kept low, the labeling efficiency of rRNA and the overall red fluorescence intensity are affected, but the nucleolar injury rate can be reduced.

Example 4:

(1) rRNA visualization stable cell line 2,3,4 acquisition, as shown in fig. 2 (d):

(1.1) rRNA cells to be marked are cultured by a mixed solution (PS) culture medium without streptomycin and are paved into a 24-well plate to form a sixth cell culture medium (the adding concentration of the cells can ensure that the cell density reaches more than 60 percent on the next day);

(1.2) on the day 24 hours after the start of the culture in step (1.1), premixing 20ng of sgRNA expression plasmid, 5ng of SpCas9 protein expression plasmid and 20ng of donor plasmid with 40ul of serum-reduced medium (Opti-medium), adding 2.4ul of Fugene transient transfection reagent (Promega) to form liposomes, and slowly adding dropwise the liposomes to the culture medium of the sixth cell to transiently transfect the cells to be labeled, wherein sgrnas to be used are sgRNA targeting ITS1, such as SEQ ID No.5, sgRNA targeting 3 ets, such as SEQ ID No.10, sgRNA targeting 5.8S, such as SEQ ID No.8, and sgRNA-sgTS 1 targeting the donor plasmid, such as SEQ ID No.11;

(1.3) after 48 hours from the completion of step (1.2), isolating the cells obtained in step (1.2) into a 96-well plate single cell clone;

(1.4) when the cells in one well of the 96-well plate had grown to the state of cell population, the single cell clones were selected by observing a red signal spot under a microscope to obtain 3' ETS rRNA stably-labeled cell line 2, ITS1rRNA stably-labeled cell line 3,5.8S rRNA stably-labeled cell line 4. The obtained cells are shown in FIG. 4 (a). In the figure, rRNA is shown in red, RPA43 (representing fiber center) is shown in green, FBL (representing dense fiber component) is shown in purple, B23 (representing particle component) is shown in blue, and the three are three components of nucleolus.

(1.5) co-localization analysis of rRNA signal and B23 protein in (a) of FIG. 4 was performed using ImageJ analysis software, and the results are shown in (B) of FIG. 4. The figure shows that the red color exhibited by rRNA can be surrounded by the blue color exhibited by B23.

The result shows that the rRNA signal point is in the nucleolus and is a real rRNA signal point.

The screening of the single cell clone is to screen the cell with rRNA mark, i.e. the cell with red signal point, or to eliminate.

The rRNA cell to be labeled is a stable cell line 1 of a visual system.

Example 5:

(1) Verification of rRNA marker specificity in rRNA stable marker cell lines:

(1.1) placing the ITS1rRNA stable labeled cells obtained in the step (1.4) in the example 4 into a confocal microscope living cell workstation, namely dynamically imaging living cells for a long time to visualize the change situation of ITS1rRNA after adding Pol I transcription inhibitor Actinomycin D for a long time: the microscope live cell workstation settings were adjusted to a temperature of 37 ℃ and to contain 5% by volume of CO ₂ 。

561 laser intensity is set to 2%, exposure time is 100ms, total shooting time is 1 hour, and shooting interval is 2 minutes. The results are shown in fig. 5 (a). In the figure, red spots of ITS1rRNA in group D, weakened or even disappeared with time by the addition of Pol I transcription inhibitor. The ITS1rRNA red highlight in the control group (no Pol I transcription inhibitor Actinomycin D added to the medium) was not significantly changed.

As shown in FIG. 5 (b), the rRNA transcriptional dynamics can be quantified by plotting the fluorescence intensity of the rRNA signal at each time point during the long-term imaging as a curve with time. As shown in the figure, the fluorescence intensity of rRNA signals in the group to which the Pol I transcription inhibitor Actinomycin D was added gradually decreased with the increase of time, while the fluorescence intensity of rRNA signals in the control group (to which the Pol I transcription inhibitor Actinomycin D was not added) did not change significantly with the increase of time.

(1.2) taking ITS1,3' ETS,5.8S rRNA stably-labeled cells obtained in the step (1.4) in example 4, putting the cells into a confocal microscope living cell workstation, namely imaging living cells for a long time to observe the change of the three rRNAs after adding a Pol I transcription inhibitor Actinomycin D30, 60 and 120 minutes: the microscope live cell workstation settings were adjusted to a temperature of 37 ℃ and contained 5% by volume CO ₂ .561 laser intensity is set to 5% and exposure time is 100ms. An image of the change with time of the fluorescence intensity of the newborn rRNA (red) signal after the addition of the Pol I transcription inhibitor Actinomycin D to 3' ETS, ITS1,5.8S rRNA was counted, and the result is shown in FIG. 5 (c).

After addition of Pol I transcription inhibitor Actinomycin D, rRNA signals were all weakened or even disappeared, indicating that rRNA in rRNA stably tagged cell lines is a true rRNA signal.

Example 6:

(1) Verifying the feasibility that the rRNA stably marked single cell clone indicates the high and low rDNA transcription level:

(1.1) on the day, culturing HEK293T cells (purchased from ATCC) by using a streptomycin-free mixed liquor (PS) culture medium and paving the HEK293T cells in T25 to form a first cell culture medium (the HEK293T cells are added at a concentration which can enable the cell density to reach more than 80% on the next day);

(1.2) the following day, lentivirus packaging was performed: premixing 2500ng shRNA virus expression plasmid (constructed by itself and used for reducing the expression of UBF, RRN3 and SRFBP1 genes), 1250ng pSPAX2 plasmid (purchased from Addgene) and 625ng PMD2.G plasmid (purchased from Addgene) by using 125ul of serum-reduced culture medium (Opti-medium), adding 15ul of Fugene transient transfection reagent (Promega) to form a liposome, dropwise adding the liposome into the supernatant of the first cell culture medium, and carrying out transient transfection on HEK293T cells in the first cell culture medium (preparing cells containing virus particles);

(1.3) after 48 hours of the transient transfection in the step (1.2), sucking the first culture medium supernatant into a 15ml centrifuge tube, adding polyethylene glycol 6000 in the volume of 1/2 of the first culture medium supernatant, and standing overnight in a4 ℃ refrigerator;

(1.4) after 48 hours of transient transfection in step (1.2), culturing 3' -ETS and ITS1rRNA stably labeled cells and plating the cells in a 24-well plate to form a second cell culture medium (the concentration of the cells to be infected is such that the next day cell density reaches about 60%);

(1.5) on the day 24 hours after the second cell culture medium is formed, taking the supernatant of the first cell culture medium obtained after polyethylene glycol 6000 is added into the 4-degree refrigerator in the step (1.3) overnight, then placing the supernatant into a 4-degree centrifuge and centrifuging the supernatant for 20 minutes at the rotating speed of 4500g, discarding the supernatant after centrifugation, keeping the precipitate, using 300ul PBS to resuspend the precipitate, and extracting the PBS containing the virus;

(1.6) preparing a PS-free culture medium containing a gene transfection enhancer (polybrene) with a concentration of 5 [ mu ] g/mL as a third culture medium to replace the second cell culture medium, so that a cell to be infected, which uses a human HeLa cell (purchased from ATCC), is cultured and transferred to the third culture medium, and then 100ul of PBS containing a virus is added dropwise to the supernatant of the third culture medium of the cell to be infected (to obtain an infected cell);

(1.7) after 12 hours of infection in step (1.6), the culture is continued by replacing fresh medium with the third medium (of infected cells), and after 48 hours, a small portion is passaged into an 8-well plate for microscopic imaging observation, and the remaining large portion is transferred to a 6-well plate for further culture. After 96 hours, carrying out microscope imaging on cells in the eight-well plate, carrying out real-time fluorescent quantitative PCR (polymerase chain reaction) detection after the cells in the six-well plate are used for extracting RNA, and detecting the change condition of rRNA and the efficiency of reducing gene expression by shRNA by using real-time fluorescent quantitative PCR (qPCR);

(1.8) taking the shRNA stable cell line cells in the eight-hole plate obtained in the step (1.7) and placing the shRNA stable cell line cells in a confocal microscope living cell workstation, so that the change conditions of 3' -ETS and ITS1rRNA after the three genes of UBF, RRN3 and SRFBP1 are reduced can be observed: the microscope live cell workstation settings were adjusted to a temperature of 37 ℃ and to contain 5% by volume of CO ₂ .561 laser intensity was set to 40%, exposure time was 100ms, cells with blue protein were cells whose gene expression was reduced by shRNA, 405 laser intensity was 80%, and exposure time was 100ms. The changes in the percentage of the neogenetic rRNA (red) signal after the expression of the 3' -ETS and ITS1 rRNA-labeled cells UBF, RRN3, SRFBP1 genes was decreased were counted, and the results are shown in (c) and (e) of FIG. 7, respectively. Statistics of 3' -ETSAnd ITS1 rRNA-labeled cells, the change in fluorescence intensity of the nascent rRNA (red) signal after the decrease in UBF, RRN3, SRFBP1 gene expression, are shown in (d) and (f) of FIG. 7, respectively. The percentage of cells containing nascent rRNA (red signal) after reduced expression of UBF, RRN3, SRFBP1 gene was reduced compared to the negative control group. The fluorescence intensity of the new rRNA (red) signal is also reduced more compared with that of the negative control (three stars of significant difference in statistical analysis represents that the difference between the two is obvious), and the expression of rRNA is reduced.

And (1.9) extracting RNA from the shRNA stable cell line in the six-hole plate obtained in the step (1.7), and then carrying out real-time fluorescent quantitative PCR (qPCR) detection. The results of the real-time fluorescent quantitative PCR (qPCR) which can detect the efficiency of reducing gene expression and the change of rRNA of shRNA after the UBF, RRN3, SRFBP1 expression is reduced are shown in (g) and (f) of fig. 7, respectively. The shRNA showed a good effect of reducing gene expression, and the rRNA was changed as in (1.8), and the expression of rRNA was reduced.

The result of the rRNA stable marked single cell indicating rDNA transcription level is consistent with the real-time fluorescent quantitative PCR (qPCR) result, and the result proves that the rRNA stable marked single cell cloning can indicate the rDNA transcription level.

Example 7:

(1) A visual stable cell line 5 was constructed as shown in fig. 2 (C):

cell line 5 was a single cell clone stably expressing stdMCP-tdTomato for U2 OS.

(1.1) on the day, culturing HEK293T cells by using a streptomycin-free mixed liquor (PS) culture medium and paving the HEK293T cells in a 12-well plate to form a first cell culture medium, wherein the HEK293T cells are added at a concentration which can enable the cell density on the next day to reach more than 80%;

(1.2) the following day, lentivirus packaging was performed: premixing 750ng of stdMCP-tdTomato virus expression plasmid, 705ng of pCMV-dR8.91 plasmid and 87ng of PMD2.G plasmid by using 75ul of serum-reduced culture medium (Opti-medium), adding 4.5ul of Fugene transient transfection reagent (Promega) to form a liposome, dropwise adding the liposome into the supernatant of the first cell culture medium, and performing transient transfection on HEK293T cells in the first cell culture medium to prepare cells containing virus particles;

(1.3) after 12 hours of transient transfection in step (1.2), aspirating the first cell culture medium supernatant and replacing the first cell culture medium with fresh medium;

(1.4) culturing the cells to be infected 48 hours after the transient transfection in the step (1.2) and paving the cells to be infected in a 24-well plate to form a second cell culture medium, wherein the adding concentration of the cells to be infected can enable the next day cell density to reach about 60%;

(1.5) on the day after 24 hours of forming the second cell culture medium, sucking the first cell culture medium supernatant obtained by replacing the fresh culture medium in the step (1.3), then placing the first cell culture medium supernatant into a centrifuge for 8 minutes at the rotating speed of 800g, taking the supernatant after centrifugation, and extracting the cell supernatant containing the virus;

(1.6) preparing a PS-free culture medium containing a gene transfection enhancer (polybrene) with the concentration of 5 mug/mL as a third culture medium to replace a second cell culture medium, so that a cell to be infected by a human U2OS cell is cultured and transferred to the third culture medium, and then 30-90 ul of cell supernatant containing the virus is taken and dripped into the third culture medium supernatant infected with the cell to be infected to obtain an infected cell;

(1.7) after 12 hours of infection in the step (1.6), replacing a fresh culture medium for a third culture medium of the infected cells, continuously culturing until the cells can be passaged to 8-well plates for microscope imaging observation, screening cell infected groups according to fluorescence intensity and infection efficiency, and separating single cell clones of 96-well plates;

the screening of the single cell clone specifically screens out cell infected groups with proper fluorescence intensity and higher infection efficiency: and selecting the cells in the single hole of the pore plate with the reserved infection efficiency of more than 30-40%, otherwise, removing the cells.

(1.8) when the cells in the single well of the 96-well plate grow to the state of cell groups, observing the fluorescence intensity under a microscope, and selecting the single-cell clone which realizes the best signal-to-noise ratio of the RNA marker.

(2) Visualization of rRNA in stable cell line 5, as shown in fig. 2 (c):

(2.1) rRNA cells to be marked are cultured by a streptomycin-free mixed solution (PS) culture medium and spread into an eight-hole chamber cover glass slide to form fourth cell culture medium cells, and the adding concentration of the fourth cell culture medium cells can enable the cell density on the next day to reach more than 40%;

(2.2) on the day 24 hours after the start of the culture in step (2.1), premixing 100ng each of the sgRNA expression plasmid targeting 5' ETS, 2.5ng of the SpCas9 protein expression plasmid and 100ng of the donor plasmid with 25ul of serum-reduced medium (Opti-medium), adding 1.5ul of Fugene transient transfection reagent (Promega) to form liposomes, slowly adding dropwise the liposomes into the supernatant of the fourth cell culture medium, and transiently transfecting the cells to be labeled with rRNA;

(2.3) after 48 hours from the completion of step (2.2), the cells obtained in step (2.2) were placed in a live microscope cell station set to a temperature of 37 ℃ and containing a volume fraction of 5% CO ₂ 。

And the microscope also comprises a microscope light source and an imaging camera, and live cell imaging visualization is carried out by setting and adjusting the laser intensity of the microscope light source and the exposure time of the imaging camera.

rRNA appeared red, i.e., in the live cell state, rRNA was observed.

The rRNA cell to be labeled in this example refers to a stable cell line 5 of a visual system.

The donor plasmid is shown in a specific schematic diagram in (a) in FIG. 2: two ends are sgTS1 recognition sequence and PAM sequence thereof, and the middle is MS2V5 _17x And (4) sequencing. The result after preparation is shown in SEQ ID No.3.

(3) Validation of rRNA labeling in U2OS cells:

(3.1) taking 5'ETS rRNA labeled cells obtained in the step (2.3) and placing the cells into a confocal microscope living cell workstation, namely dynamically imaging living cells for a long time to visualize the change condition of 5' ETS rRNA after adding Pol I transcription inhibitor Actinomycin D for a long time: the microscope live cell workstation settings were adjusted to a temperature of 37 ℃ and contained 5% by volume CO ₂ 。

561 laser intensity is set to 5%, exposure time is 100ms, total shooting time is 2 hours, and shooting interval is 2 minutes. The results are shown in FIG. 6 (a). In the figure, the red bright spot of the ETS rRNA group 5' added with Pol I transcription inhibitor Actinomycin D weakens or even disappears as time goes on. Whereas the 5' ETS rRNA red highlight in Actinomycin D, a Pol I transcription inhibitor, was not added to the control group medium, and did not change significantly.

As shown in FIG. 6 (b), the fluorescence intensity of rRNA signal at each time point during the long-term imaging is plotted as a line graph which changes with time, and the transcription kinetics of rRNA can be quantified. As shown in the figure, the fluorescence intensity of rRNA signal in the group D to which the Pol I transcription inhibitor Actinomycin was added gradually decreased with the time, while the fluorescence intensity of rRNA signal in the control group medium to which the Pol I transcription inhibitor Actinomycin D was not added did not change significantly with the time.

After addition of Pol I transcription inhibitor Actinomycin D, rRNA signals were all weakened or even disappeared, indicating that rRNA in U2OS cells is a true rRNA signal.

Example 8:

(1) A visual stable cell line 6 was constructed as shown in fig. 2 (b):

cell line 6 is a single cell clone stably expressing stdMCP-tdTomato in RPE-1 cells.

(1.1) on the day, culturing HEK293T cells by using a streptomycin-free mixed liquor (PS) culture medium and paving the HEK293T cells in a 12-well plate to form a first cell culture medium, wherein the HEK293T cells are added at a concentration which can enable the cell density on the next day to reach more than 80%;

(1.2) the following day, lentivirus packaging was performed: premixing 750ng of stdMCP-tdTomato virus expression plasmid, 705ng of pCMV-dR8.91 plasmid and 87ng of PMD2.G plasmid by using 75ul of serum-reduced culture medium (Opti-medium), adding 4.5ul of Fugene transient transfection reagent (Promega) to form a liposome, dropwise adding the liposome into the supernatant of the first cell culture medium, and performing transient transfection on HEK293T cells in the first cell culture medium to prepare cells containing virus particles;

(1.3) after 12 hours of transient transfection in step (1.2), aspirating the first cell culture medium supernatant and replacing the first cell culture medium with fresh medium;

(1.4) culturing the cells to be infected 48 hours after the transient transfection in the step (1.2) and paving the cells to be infected in a 24-well plate to form a second cell culture medium, wherein the adding concentration of the cells to be infected can enable the cell density to reach about 60% in the next day;

(1.5) on the day 24 hours after the second cell culture medium is formed, sucking the first cell culture medium supernatant obtained by replacing the fresh culture medium in the step (1.3), then placing the first cell culture medium supernatant into a centrifuge for 8 minutes at the rotation speed of 800g, taking the supernatant after centrifugation, and extracting a cell supernatant containing viruses;

(1.6) preparing a PS-free culture medium containing a gene transfection enhancer (polybrene) with the concentration of 5 mu g/mL as a third culture medium to replace a second cell culture medium, transferring the cells to be infected by adopting human RPE-1 cells to the third culture medium, and then taking 30-90 ul of cell supernatant containing the virus to be dropwise added into the third culture medium supernatant infected with the cells to be infected to obtain infected cells;

(1.7) after 12 hours of infection in the step (1.6), replacing a fresh culture medium for the third culture medium of the infected cells, continuing culturing until the third culture medium can be passaged to an 8-well plate for microscope imaging observation, screening a cell infected population according to the fluorescence intensity and the infection efficiency, and separating single cell clones of a 96-well plate;

the screening of the single cell clone specifically screens out cell infected groups with proper fluorescence intensity and higher infection efficiency: and selecting the cells in the single hole of the pore plate with the preserved infection efficiency of more than 30-40%, otherwise, removing.

(1.8) when the cells in the single well of the 96-well plate grow to the state of cell groups, observing the fluorescence intensity under a microscope, and selecting the single-cell clone with the best RNA mark signal-to-noise ratio.

(2) Visualization of rRNA in stable cell line 6, as shown in fig. 2 (c):

(2.1) rRNA cells to be marked are cultured by a streptomycin-free mixed solution (PS) culture medium and spread into an eight-hole chamber cover glass slide to form fourth cell culture medium cells, and the adding concentration of the fourth cell culture medium cells can enable the cell density on the next day to reach more than 40%;

(2.2) on the day after 24 hours from the start of the culture in step (2.1), premixing 100ng each of the sgRNA expression plasmid targeting 5' ETS, 2.5ng SpCas9 protein expression plasmid and 100ng of the donor plasmid with 25ul of serum-reduced medium (Opti-medium), adding 1.5ul of Fugene transient transfection reagent (Promega) to form liposomes, slowly adding the liposomes dropwise to the supernatant of the fourth cell culture medium, and transiently transfecting the rRNA cells to be labeled;

(2.3) after 48 hours from the completion of step (2.2), the cells obtained in step (2.2) were placed in a live microscope cell station set to a temperature of 37 ℃ and containing a volume fraction of 5% CO ₂ 。

And the microscope also comprises a microscope light source and an imaging camera, and live cell imaging visualization is carried out by setting and adjusting the laser intensity of the microscope light source and the exposure time of the imaging camera.

rRNA appeared red, i.e., in the live cell state, rRNA was observed.

The rRNA cell to be labeled in this example refers to the stable cell line 6 of the visual system.

The donor plasmid is shown in a specific schematic diagram in (b) in FIG. 2: two ends are sgTS1 recognition sequence and PAM sequence thereof, and the middle is MS2V5 _17x And (4) sequencing. The result after preparation is shown in SEQ ID No.3.

(3) Validation of rRNA markers in RPE-1 cells:

(3.1) taking 5'ETS rRNA labeled cells obtained in the step (2.3) and placing the cells into a confocal microscope living cell workstation, namely dynamically imaging living cells for a long time to visualize the change condition of 5' ETS rRNA after adding Pol I transcription inhibitor Actinomycin D for a long time: the microscope live cell workstation settings were adjusted to a temperature of 37 ℃ and contained 5% by volume CO ₂ 。

561 laser intensity is set to 5%, exposure time is 100ms, total shooting time is 2 hours, and shooting interval is 2 minutes. The results are shown in FIG. 6 (c). In the figure, the red bright spot of the ETS rRNA group 5' added with Pol I transcription inhibitor Actinomycin D weakens or even disappears as time goes on. Whereas the 5' ETS rRNA red highlight in Actinomycin D, a Pol I transcription inhibitor, was not added to the control group medium, and did not change significantly.

As shown in FIG. 6 (d), the fluorescence intensity of rRNA signal at each time point during the long-term imaging was plotted as a line graph with time, and the transcription kinetics of rRNA was quantified. As shown in the figure, the fluorescence intensity of rRNA signal in the group D to which the Pol I transcription inhibitor Actinomycin was added gradually decreased with the time, while the fluorescence intensity of rRNA signal in the control group medium to which the Pol I transcription inhibitor Actinomycin D was not added did not change significantly with the time.

After addition of Pol I transcription inhibitor Actinomycin D, the rRNA signals were all weakened or even disappeared, indicating that the rRNA in RPE-1 cells was the true rRNA signal.

Example 9:

after obtaining a stable marker of rRNA in living cells, a small anti-tumor drug was added to observe changes in rRNA, as shown in fig. 2 (e):

(1) Knowing the working concentration and working time of the known antitumor small molecule drugs, generally the quantity concentration of a substance;

(2) Preparing a fifth culture medium containing the anti-tumor small molecule drugs, wherein the substance quantity concentration of the anti-tumor small molecule drugs in the fifth culture medium should reach the working concentration;

(3) Taking the 3' ETS rRNA stably-labeled cell line 2, the ITS1rRNA stably-labeled cell line 3, the 5.8S rRNA stably-labeled cell line 4 obtained in the step (1.4) of the example 4, sucking the supernatant of the fourth cell culture medium, and replacing the fourth cell culture medium with a fifth culture medium containing the antitumor small molecule medicament;

(4) After a specified time of completion of step (3) (depending on the onset time of the anti-tumor small molecule drug provided), the cells obtained in step (3) are placed in a microscope live cell workstation set to a temperature of 37 ℃ and containing 5% by volume of CO ₂ 。

The microscope comprises a microscope light source and an imaging camera, and living cells are imaged and visualized by setting and adjusting the laser intensity of the microscope light source and the exposure time of the imaging camera.

As shown in fig. 8 (a), the red spots of the rRNA in the whole cells of the group added with the anti-tumor small molecule drugs are weakened or even disappeared compared to the control group (the anti-tumor small molecule drugs are not added to the culture medium).

As shown in FIG. 8 (b), the total fluorescence intensity of ribosomal RNA signals in the group containing the small anti-tumor drug was significantly decreased compared to the total fluorescence intensity of rRNA signals in the control group (the medium was not supplemented with the small anti-tumor drug).

The result shows that a stable cell line with stable and visible rRNA in living cells can be obtained, and the cell line can also be used for directly observing the influence of the anti-tumor small molecules on the rRNA by a real-time imaging method, so that the influence of the anti-tumor small molecule drugs on the rRNA can be more intuitively and accurately known.

Therefore, the rRNA is visualized in living cells, the influence of the anti-tumor small molecules on the rRNA is directly observed by a real-time imaging method, and the method is convenient and quick.

Example 10:

(1) Transient visualization of rRNA in various regions of stable cell line 1:

(1.1) As in the steps (2.1) to (2.2) of example 2, 5' ETS,18S,28S,5.8S, ITS1, ITS2,3' ETS regions were inserted with donor plasmids and expressed, wherein sgRNAs to be used were sgRNAs targeting the 5' ETS region, respectively, as shown in SEQ ID No. 4; the sgRNA of the targeted 18S region is shown as SEQ ID No. 7; the sgRNA of a 28S-targeted region is shown as SEQ ID No. 9; the sgRNA targeting the 5.8S region is shown as SEQ ID No. 8; the sgRNA of the target ITS1 region is shown in SEQ ID No. 5; the sgRNA targeting the ITS2 region is shown in SEQ ID No. 6; the sgRNA of the target 3' ETS region is shown as SEQ ID NO. 10; sgRNA- - -sgTS1 of the targeting donor plasmid is shown as SEQ ID No. 11.

(2) And (3) visualizing rRNA of each region and adding an anti-tumor small molecule drug CX-5461 for live cell imaging:

(2.1) knowing the working concentration and working time of the known anti-tumor small molecule drug CX-5461, wherein the working concentration and working time are usually the quantity concentration of a substance;

(2.2) preparing a fifth culture medium containing the anti-tumor small-molecule medicine (the concentration of the anti-tumor small-molecule medicine in the fifth culture medium should reach the working concentration);

(2.3) taking the cells with the red bright spots seen in the step (1), sucking the supernatant of the fourth cell culture medium, and replacing the fourth cell culture medium with a fifth cell culture medium containing an anti-tumor small molecule drug CX-5461;

(2.4) placing the cells obtained 2 hours after the completion of the step (2.3) into a live cell workstation of a wide-field microscope, namely, imaging the live cells for a long time to visualize the change condition of rRNA in each region after the antitumor small molecule medicament CX-5461 is added: the microscope live cell workstation settings were adjusted to a temperature of 37 ℃ and to contain 5% by volume of CO ₂ 。

The TEX RED fluorescence intensity was set at 5%, the exposure time was 100ms, and the imaging mode was Binning 2X 2. As shown in fig. 9 (b), the overall fluorescence intensity of rRNA signals in different regions of the group containing the anti-tumor small molecule drug CX-5461 was significantly decreased compared to the overall fluorescence intensity of rRNA signals in the control group (the anti-tumor small molecule drug was not added to the medium).

The result shows that the influence of the antitumor small molecules on the rRNA in different areas can be directly and accurately known by visualizing the rRNA in different areas in living cells and directly observing the influence of the antitumor small molecules on the rRNA in different areas by a real-time imaging method.

Example 11:

after obtaining a stable marker of rRNA in living cells, the antitumor small molecule drug 5-Fluorouracil was added to observe the change in rRNA, as shown in fig. 2 (e):

(1) Knowing the working concentration and working time of the known antitumor small molecule drugs, generally the quantity concentration of a substance;

(2) Preparing a seventh culture medium containing the anti-tumor small molecule drug 5-fluoroauracil, wherein the quantity concentration of the anti-cancer small molecule drug in the seventh culture medium is required to reach the working concentration;

(3) Taking the 3' ETS rRNA stable marker cell line 2 obtained in the step (1.4) of example 4, the ITS1rRNA stable marker cell line 3,5.8S rRNA stable marker cell line 4, sucking the supernatant of the fourth cell culture medium, and replacing the fourth cell culture medium with a seventh culture medium containing 5-Fluorocouracil serving as an antitumor small molecule drug;

(4) After the specified time of the step (3) is completed (according to the onset time of the provided antitumor small molecule drugs), the cells obtained in the step (3) are treatedPlacing in a microscope living cell workstation, adjusting the microscope living cell workstation to 37 deg.C and containing 5% by volume of CO ₂ 。

The microscope comprises a microscope light source and an imaging camera, and living cells are imaged and visualized by setting and adjusting the laser intensity of the microscope light source and the exposure time of the imaging camera.

As shown in FIG. 10 (a), in the figure, 3' ETS rRNA-stable marker cells and ITS1 rRNA-stable marker cells in the group of 5-fluorooracic cells added with the anti-tumor small molecule drug showed stronger red spots than in the control group (the anti-tumor small molecule drug was not added to the medium).

As shown in FIG. 10 (b), the ratio of rRNA-containing red signal point cells in 3' ETS rRNA-stably labeled cells and ITS1 rRNA-stably labeled cells in the group of 5-fluorooracic cells to the group of ITS1 rRNA-stably labeled cells was increased as compared with the control group (the medium was not supplemented with the anti-tumor small molecule drug).

As shown in FIG. 10 (c), the total fluorescence intensity of rRNA signals in 5-fluoroouracil group, 3' ETS rRNA stably labeled cells and ITS1rRNA stably labeled cells, to which antitumor small molecule drug was added, was increased as compared with the control group (to which antitumor small molecule drug was not added to the medium).

The results showed that after inhibition of pre-rRNA cleavage, the nascent rRNA signal (3' ETS, ITS1 rRNA) accumulated, whereas the mature rRNA (5.8S) did not change significantly.

Therefore, the rRNA is visualized in living cells, the influence of the anti-tumor small molecules on the rRNA is directly observed by a real-time imaging method, and the method is convenient and quick.

Example 12:

after obtaining a stable marker of rRNA in living cells, after starvation treatment of the cells, nutrient serum is added, and changes of the rRNA are observed:

(1) rRNA cell to be marked is cultured with mixed liquid (PS) culture medium without streptomycin and spread to six-hole plate to form the eighth cell culture medium with the adding concentration of the cell, which can make the next day cell density reach more than 60%;

(2) Replacing the eighth cell culture medium with a serum-free ninth cell culture medium on the day 24 hours after the start of the culture of step (1);

(3) Replacing the ninth medium without serum with a tenth cell culture medium with nutrient serum 24 hours after completion of step (2);

(4) After 30 minutes, the cells obtained in step (3) were placed in a microscope live cell workstation set to a temperature of 37 ℃ and containing 5% by volume of CO ₂ 。

And the microscope also comprises a microscope light source and an imaging camera, and live cell imaging visualization is carried out by setting and adjusting the laser intensity of the microscope light source and the exposure time of the imaging camera.

As a result, as shown in FIG. 11 (a), the rRNA stable marker cells showed an enhanced red spot in rRNA as a whole compared with the control group (in which no nutrient serum was added to the medium) in which the nutrient serum group was added.

As shown in fig. 11 (b), when the nutrient serum group was added, the total fluorescence intensity of rRNA signals in rRNA-stably labeled cells was increased as compared with the control group (nutrient serum was not added to the medium).

The results show that when the external environment stimulates, for example, the cells are starved and then are given with nutrient serum, the rRNA transcription can be promoted, and the phenomenon can be well reflected by our system.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Zhejiang university

<120> live cell ribosomal RNA visualization system and application thereof

<130> P210111

<160> 16

<170> PatentIn version 3.5

<210> 1

<211> 918

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 1

gaaggtgaca accggtaacc tacaaacggg tggaggatca ccccacccga cacttcacaa 60

tcaaggggta caatacacaa gggtggagga acaccccacc ctccagacac attacacaga 120

aatccaatca aacagaagca ccatcagggc ttctgctacc aaatttatct caaaaaacta 180

caacaaggaa tcaccatcag ggattccctg tgcaatatac gtcaaacgag ggccacgacg 240

ggaggacgat cacgcctccc gaatatcggc atgtctggct ttcgaattca gtgcgtggag 300

catcagccca cgcagccaat cagagtcgaa tacaagtcga ctttcgcgaa gagcatcagc 360

cttcgcgcca ttcttacaca aaccacactc tcccctacag gaacagcatc agcgttcctg 420

cccagtaccc aactcaagaa aatttatgtc cccatgcagc atcagcgcat gggccccaag 480

aatacatccc caacaaaatc acatccgagc accaacaggg ctcggagtgt tgtttcttgt 540

ccaactggac aaaccctcca tggaccatca ggccatggac tctcaccaac aagacaaaaa 600

ctactcttct cgaagcagca tcagcgcttc gaaacactcg agcatacatt gtgcctattt 660

cttgggtgga cgatcacgcc acccatgctc tcacgaattt caaaacacgg acaaggacga 720

gcaccaccag ggctcgtcgt tccacgtcca atacgattac ttacctttcg ggatcacgat 780

cacggatccc gcagctacat cacttccact caggacattc aagcatgcac gatcacggca 840

tgctccacaa gtctcaacca cagaaactac caaatgggtt cagcaccagc gaacccactc 900

ctacctcaaa cctcttcc 918

<210> 2

<211> 2031

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 2

gcaccgatgc tctccgagga ggaattcttc gctcgctcgt tcgttcgccg cccggccccg 60

ccgccgcgag agccgagaac tcgggaggga gacggggggg agagagagag agagagagag 120

agagagagag agagagagag aaagaagggc gtgtcgttgg tgtgcgcgtg tcgtggggcc 180

ggcgggcggc ggggagcggt ccccggccgc ggccccgacg acgtgggtgt cggcgggcgc 240

gggggcggtt ctcggcggcg tcgcggcggg tctggggggg tctcggtgcc ctcctccccg 300

ccggggcccg tcgtccggcc ccgccgcgcc ggctccccgt cttcggggcc ggccggattc 360

ccgtcgcctc cgccgcgccg ctccgcgccg ccgggcacgg ccccgctcgc tctccccggc 420

cttcccgcta gggcgtctcg agggtcgggg gccggacgcc ggtcccctcc cccgcctcct 480

cgtccgcccc cccgccgtcc aggtacctag cgcgttccgg cgcggaggtt taaagacccc 540

ttggggggat cgcccgtccg cccgtgggtc gggggcggtg gtgggcccgc gggggagtcc 600

cgtcgggagg ggcccggccc ctcccgcgcc tccaccgcgg actccgctcc ccggccgggg 660

ccgcgccgcc gccgccgccg cggcggccgt cgggtggggg aagcttgaag gtgacaaccg 720

gtaacctaca aacgggtgga ggatcacccc acccgacact tcacaatcaa ggggtacaat 780

acacaagggt ggaggaacac cccaccctcc agacacatta cacagaaatc caatcaaaca 840

gaagcaccat cagggcttct gctaccaaat ttatctcaaa aaactacaac aaggaatcac 900

catcagggat tccctgtgca atatacgtca aacgagggcc acgacgggag gacgatcacg 960

cctcccgaat atcggcatgt ctggctttcg aattcagtgc gtggagcatc agcccacgca 1020

gccaatcaga gtcgaataca agtcgacttt cgcgaagagc atcagccttc gcgccattct 1080

tacacaaacc acactctccc ctacaggaac agcatcagcg ttcctgccca gtacccaact 1140

caagaaaatt tatgtcccca tgcagcatca gcgcatgggc cccaagaata catccccaac 1200

aaaatcacat ccgagcacca acagggctcg gagtgttgtt tcttgtccaa ctggacaaac 1260

cctccatgga ccatcaggcc atggactctc accaacaaga caaaaactac tcttctcgaa 1320

gcagcatcag cgcttcgaaa cactcgagca tacattgtgc ctatttcttg ggtggacgat 1380

cacgccaccc atgctctcac gaatttcaaa acacggacaa ggacgagcac caccagggct 1440

cgtcgttcca cgtccaatac gattacttac ctttcgggat cacgatcacg gatcccgcag 1500

ctacatcact tccactcagg acattcaagc atgcacgatc acggcatgct ccacaagtct 1560

caaccacaga aactaccaaa tgggttcagc accagcgaac ccactcctac ctcaaacctc 1620

ttcctctaga ctttacccgg cggccgtcgc gcgcctgccg cgcgtgtggc gtgcgccccg 1680

cgccgtgggg gcgggaaccc ccgggcgcct gtggggtggt gtccgcgctc gcccccgcgt 1740

gggcggcgcg cgcctccccg tggtgtgaaa ccttccgacc cctctccgga gtccggtccc 1800

gtttgctgtc tcgtctggcc ggcctgaggc aaccccctct cctcttgggc ggggggggcg 1860

gggggacgtg ccgcgccagg aagggcctcc tcccggtgcg tcgtcgggag cgccctcgcc 1920

aaatcgacct cgtacgactc ttagcggtgg atcactcggc tcgtgcgtcg atgaagaacg 1980

cagctagctg cgagaattaa tgtgatatcg caccgatgct ctccgaggag g 2031

<210> 3

<211> 962

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 3

gcaccgatgc tctccgagga gggaaggtga caaccggtaa cctacaaacg ggtggaggat 60

caccccaccc gacacttcac aatcaagggg tacaatacac aagggtggag gaacacccca 120

ccctccagac acattacaca gaaatccaat caaacagaag caccatcagg gcttctgcta 180

ccaaatttat ctcaaaaaac tacaacaagg aatcaccatc agggattccc tgtgcaatat 240

acgtcaaacg agggccacga cgggaggacg atcacgcctc ccgaatatcg gcatgtctgg 300

ctttcgaatt cagtgcgtgg agcatcagcc cacgcagcca atcagagtcg aatacaagtc 360

gactttcgcg aagagcatca gccttcgcgc cattcttaca caaaccacac tctcccctac 420

aggaacagca tcagcgttcc tgcccagtac ccaactcaag aaaatttatg tccccatgca 480

gcatcagcgc atgggcccca agaatacatc cccaacaaaa tcacatccga gcaccaacag 540

ggctcggagt gttgtttctt gtccaactgg acaaaccctc catggaccat caggccatgg 600

actctcacca acaagacaaa aactactctt ctcgaagcag catcagcgct tcgaaacact 660

cgagcataca ttgtgcctat ttcttgggtg gacgatcacg ccacccatgc tctcacgaat 720

ttcaaaacac ggacaaggac gagcaccacc agggctcgtc gttccacgtc caatacgatt 780

acttaccttt cgggatcacg atcacggatc ccgcagctac atcacttcca ctcaggacat 840

tcaagcatgc acgatcacgg catgctccac aagtctcaac cacagaaact accaaatggg 900

ttcagcacca gcgaacccac tcctacctca aacctcttcc gcaccgatgc tctccgagga 960

gg 962

<210> 4

<211> 22

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

acacgcacgg cacggagcca gc 22

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 5

cgggtggggg ctttacccgg 20

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 6

cgtcccgagc ttccgcgtcg 20

<210> 7

<211> 19

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 7

cgacccgggg aggtagtga 19

<210> 8

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 8

cgacacttcg aacgcacttg 20

<210> 9

<211> 19

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 9

cggccgaggt gggatcccg 19

<210> 10

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 10

ggagcgtggt ttgggagccg 20

<210> 11

<211> 19

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 11

gcaccgatgc tctccgagg 19

<210> 12

<211> 1437

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 12

atggcttcta actttactca gttcgttctc gtcgacaatg gcggaactgg cgacgtgact 60

gtcgccccaa gcaacttcgc taacgggatc gctgaatgga tcagctctaa ctcgcgttca 120

caggcttaca aagtaacctg tagcgttcgt cagagctctg cgcagaatcg caaatacacc 180

atcaaagtcg aggtgcctaa aggcgcctgg cgttcgtact taaatatgga actaaccatt 240

ccaattttcg ccacgaattc cgactgcgag cttattgtta aggcaatgca aggtctccta 300

aaagatggaa acccgattcc ctcagcaatc gcagcaaact ccggcatcta cgccatggct 360

tctaacttta ctcagttcgt tctcgtcgac aatggcggaa ctggcgacgt gactgtcgcc 420

ccaagcaact tcgctaacgg gatcgctgaa tggatcagct ctaactcgcg ttcacaggct 480

tacaaagtaa cctgtagcgt tcgtcagagc tctgcgcaga atcgcaaata caccatcaaa 540

gtcgaggtgc ctaaaggcgc ctggcgttcg tacttaaata tggaactaac cattccaatt 600

ttcgccacga attccgactg cgagcttatt gttaaggcaa tgcaaggtct cctaaaagat 660

ggaaacccga ttccctcagc aatcgcagca aactccggca tctacgcgga ttctagaatg 720

gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc 780

gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc 840

aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc 900

gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag 960

cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc 1020

aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg 1080

aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag 1140

ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc 1200

atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac 1260

cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac 1320

ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg 1380

ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaa 1437

<210> 13

<211> 2214

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 13

atgggcccaa aaaagaaaag aaaagttggc tacccctacg acgtgcccga ctacgccatc 60

gaaggccgcc atatgctagc cgcttctaac tttactcagt tcgttctcgt cgacaatggc 120

ggaactggcg acgtgactgt cgccccaagc aacttcgcta acgggatcgc tgaatggatc 180

agctctaact cgcgttcaca ggcttacaaa gtaacctgta gcgttcgtca gagctctgcg 240

cagaatcgca aatacaccat caaagtcgag gtgcctaaag gcgcctggcg ttcgtactta 300

aatatggaac taaccattcc aattttcgcc acgaattccg actgcgagct tattgttaag 360

gcaatgcaag gtctcctaaa agatggaaac ccgattccct cagcaatcgc agcaaactcc 420

ggcatctacg ccatggccag caacttcacc cagttcgtgc tggtggacaa cggcggcacc 480

ggcgacgtga ccgtggcccc cagcaacttc gccaacggca tcgccgagtg gatcagcagc 540

aacagcagaa gccaggccta caaggtgacc tgcagcgtga gacagagcag cgcccagaac 600

agaaagtaca ccatcaaggt ggaggtgccc aagggcgcct ggagaagcta cctgaacatg 660

gagctgacca tccccatctt cgccaccaac agcgactgcg agctgatcgt gaaggccatg 720

cagggcctgc tgaaggacgg caaccccatc cccagcgcca tcgccgccaa cagcgattct 780

agaatggtga gcaagggcga ggaggtcatc aaagagttca tgcgcttcaa ggtgcgcatg 840

gagggctcca tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac 900

gagggcaccc agaccgccaa gctgaaggtg accaagggcg gccccctgcc cttcgcctgg 960

gacatcctgt ccccccagtt catgtacggc tccaaggcgt acgtgaagca ccccgccgac 1020

atccccgatt acaagaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac 1080

ttcgaggacg gcggtctggt gaccgtgacc caggactcct ccctgcagga cggcacgctg 1140

atctacaagg tgaagatgcg cggcaccaac ttcccccccg acggccccgt aatgcagaag 1200

aagaccatgg gctgggaggc ctccaccgag cgcctgtacc cccgcgacgg cgtgctgaag 1260

ggcgagatcc accaggccct gaagctgaag gacggcggcc actacctggt ggagttcaag 1320

accatctaca tggccaagaa gcccgtgcaa ctgcccggct actactacgt ggacaccaag 1380

ctggacatca cctcccacaa cgaggactac accatcgtgg aacagtacga gcgctccgag 1440

ggccgccacc acctgttcct ggggcatggc accggcagca ccggcagcgg cagctccggc 1500

accgcctcct ccgaggacaa caacatggcc gtcatcaaag agttcatgcg cttcaaggtg 1560

cgcatggagg gctccatgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc 1620

ccctacgagg gcacccagac cgccaagctg aaggtgacca agggcggccc cctgcccttc 1680

gcctgggaca tcctgtcccc ccagttcatg tacggctcca aggcgtacgt gaagcacccc 1740

gccgacatcc ccgattacaa gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg 1800

atgaacttcg aggacggcgg tctggtgacc gtgacccagg actcctccct gcaggacggc 1860

acgctgatct acaaggtgaa gatgcgcggc accaacttcc cccccgacgg ccccgtaatg 1920

cagaagaaga ccatgggctg ggaggcctcc accgagcgcc tgtacccccg cgacggcgtg 1980

ctgaagggcg agatccacca ggccctgaag ctgaaggacg gcggccacta cctggtggag 2040

ttcaagacca tctacatggc caagaagccc gtgcaactgc ccggctacta ctacgtggac 2100

accaagctgg acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgagcgc 2160

tccgagggcc gccaccacct gttcctgtac ggcatggacg agctgtacaa gtaa 2214

<210> 14

<211> 22

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 14

gcaccgatgc tctccgagga gg 22

<210> 15

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 15

ggagcagacg atatggcgtc gctcc 25

<210> 16

<211> 19

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 16

gggccctgaa gaagggccc 19

Claims (10)

1. A genetically engineered cell having incorporated into its rDNA an exogenous DNA sequence encoding an RNA marker sequence that becomes part of rRNA upon rDNA transcription.

2. The genetically engineered cell of claim 1, further expressing an RNA binding protein that specifically binds to the RNA marker sequence.

3. The genetically engineered cell of claim 2, wherein the RNA binding protein is linked to a reporter molecule; preferably, the reporter molecule is a fluorescent protein or fluorescein.

4. A nucleic acid construct for gene knock-in, the structure of which is shown in formula I or formula II,

S-M-S, formula I

S-5'HA-M-3' HA-S, formula II

In the formula I, S is a target sequence which can be recognized and sheared by Cas9 protein, and M is a donor sequence; each "-" is a bond or a connecting sequence;

in formula II, S is a target sequence which can be recognized and cut by Cas9, and M is a donor sequence; 5'HA is the homologous arm 5' of the genomic target site into which the donor sequence is to be inserted, 3'HA is the homologous arm 3' of the genomic target site into which the donor sequence is to be inserted, each "-" is a bond or a linking sequence.

5. An expression cassette comprising the nucleic acid construct of claim 4; preferably, the expression cassette is cleaved intracellularly by Cas9 to release the fragmented M sequence as a recombinant donor template.

6. An expression vector comprising the expression cassette of claim 4 or the nucleic acid construct of the second aspect of the invention; preferably, the expression vector is a plasmid, or a viral vector.

7. A kit for preparing a genetically engineered cell, comprising the expression vector of claim 6, the expression cassette of claim 5, the nucleic acid construct of claim 4; preferably, the kit further comprises a Cas9 protein expression plasmid; more preferably, the kit further includes a first sgRNA expression plasmid that targets the S element of the nucleic acid construct; most preferably, the kit further includes a second sgRNA expression plasmid, the second sgRNA targeting rDNA.

8. Use of the genetically engineered cell of claim 1 in screening for a drug.

9. A method for screening compounds based on live cell imaging, said method comprising the steps of:

(i) Contacting a compound to be screened with the genetically engineered cell of claim 1, and

(ii) And observing the fluorescence intensity indicating the expression quantity of rRNA in the cell nucleus of the genetically engineered cell.

10. A method of producing a genetically engineered cell, said method comprising the steps of:

(i) Providing a first genetically engineered cell capable of stably expressing the fusion protein, and

(ii) Inserting an exogenous donor DNA capable of transcribing an RNA marker sequence into the rDNA of the first genetically engineered cell;

wherein the fusion protein is an RNA binding protein-fluorescent protein fusion protein;

the exogenous donor DNA can express an RNA marker sequence along with rDNA transcription; and, the RNA binding protein specifically recognizes the RNA marker sequence.

Images