Non-coding RNA combination for inducing totipotency of mouse embryonic stem cells and application thereof

Abstract

The application provides a non-coding RNA combination for inducing totipotency of mouse embryonic stem cells and application thereof, and relates to the technical field of biology.The combination comprises: an enhancer RNA with a sequence shown as SEQ ID NO:1 or SEQ ID NO:2, and a long-chain non-coding RNA with a sequence shown as SEQ ID NO:3.The application discloses, for the first time, that the CRISPR activation system is used to synergistically up-regulate the expression of the combination, and the conversion of mouse embryonic stem cells to two-cell-like cells can be efficiently promoted.The application does not need to integrate exogenous transcription factors, and thus the risk of genome is avoided; the sgRNA is precisely targeted, and thus the defect of non-specific off-target of chemical small molecules is overcome; a significant synergistic effect is generated, the induction efficiency of two-cell-like cells is significantly improved to about 11.6%, and a new strategy which is safer, more efficient and controllable is provided for stem cell fate reprogramming and early development research.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a combination of non-coding RNAs that induce pluripotency in mouse embryonic stem cells and their applications. Background Technology

[0002] Mouse embryonic stem cells (mESCs) are a type of pluripotent stem cells derived from the inner cell mass of mouse blastocysts. They possess pluripotency, enabling self-renewal and the potential to differentiate into all cell types of the three germ layers (endoderm, mesoderm, and ectoderm), but they cannot contribute to extraembryonic tissues. In contrast, embryonic cells from mouse fertilized eggs developing to the two-cell stage possess totipotency, capable of developing into a complete individual (including all embryonic and extraembryonic tissues). Notably, researchers have discovered a rare (approximately 0.1%-1%) transient cell population within the in vitro cultured mESC population that highly mimics the two-cell embryo in both transcriptome and epigenome—the two-cell-like cells (2CLCs). This cell subpopulation provides a valuable in vitro model for studying early embryonic development, mechanisms regulating cell totipotency, and developing novel regenerative medicine strategies.

[0003] To utilize 2CLCs cell models, the current methods for inducing or enriching 2CLCs mainly rely on the following: (1) Overexpression of exogenous transcription factors: For example, overexpression of transcription factors Dux, DPPA2 / 4, etc., can effectively drive mESCs to transition to the 2CLC state. However, such methods usually require the integration of exogenous genes into the cell genome, which has potential problems such as insertion mutations, oncogenic risks, and uncontrollable gene expression, which seriously limits its safety and feasibility in clinical applications such as cell therapy; (2) Chemical small molecule treatment: Epigenetic modification drugs such as DNA methylation inhibitors (such as 5-azacytidine) and histone deacetylase inhibitors can increase the proportion of 2CLCs to a certain extent. However, the mechanism of action of chemical small molecules usually lacks gene specificity, which can cause extensive epigenetic and transcriptomic changes throughout the genome, resulting in significant off-target effects and unpredictable cell state disturbances, and the induction efficiency and specificity are not ideal; (3) Single endogenous factor manipulation: In recent years, the role of endogenous non-coding RNA in cell fate regulation has been gradually recognized. However, existing research largely focuses on the functional analysis of single non-coding RNAs, lacking a systematic exploration of the synergistic regulation of totipotent networks by "combinations of non-coding RNAs." In other words, the specific functions and synergistic effects of non-coding RNAs, especially eRNAs, in establishing and maintaining totipotent networks remain poorly understood. Due to their functional redundancy and the complexity of their mechanisms of action, studying their function through traditional methods such as gene overexpression often faces challenges.

[0004] Based on the current state of research, we offer a novel approach to address the aforementioned issues. Summary of the Invention

[0005] The purpose of this invention is to provide a combination of non-coding RNAs for inducing pluripotency in mouse embryonic stem cells and its application. Addressing the problems of low efficiency, insufficient specificity, and poor safety in existing methods for inducing embryonic stem cell pluripotency, as well as the lack of understanding of the crucial role of endogenous non-coding RNAs, especially their synergistic effects, in pluripotency regulation, this invention aims to provide a novel strategy for inducing pluripotent stem cells that is targeted, controllable, safer, and more efficient.

[0006] In a first aspect, the present invention provides a non-coding RNA combination for inducing pluripotency of mouse embryonic stem cells, comprising:

[0007] (1) The enhancer RNA with the sequence shown in SEQ ID NO:1 is named ENH323047;

[0008] (2) The enhancer RNA with the sequence shown in SEQ ID NO:2 is named ENH264069;

[0009] (3) The long non-coding RNA with the sequence shown in SEQ ID NO:3 is named ENSMUSG00000106683.

[0010] Secondly, the present invention provides an application of the above-mentioned non-coding RNA combination in inducing mouse embryonic stem cells to transform into a pluripotent two-cell-like cell state, which is achieved by synergistically upregulating the expression levels of three RNAs in the above-mentioned non-coding RNA combination.

[0011] Optionally, the application is achieved by employing a CRISPR activation system to endogenously and synergistically activate the expression of three RNAs in a combination of non-coding RNAs.

[0012] Optionally, the CRISPR activation system includes guide RNAs that target the respective RNA regulatory regions in the combination of non-coding RNAs, the guide RNAs comprising:

[0013] (1) sgRNAs1 targeting the sequence regulatory region shown in SEQ ID NO:1, including SEQ ID NO:4 to SEQ ID NO:12;

[0014] (2) sgRNAs2 targeting the sequence regulatory region shown in SEQ ID NO:2, including SEQ ID NO:13 to SEQ ID NO:25;

[0015] (3) sgRNAs3 targeting the sequence regulatory region shown in SEQ ID NO:3, including SEQ ID NO:26 to SEQ ID NO:31.

[0016] Optionally, the CRISPR activation system further includes the dCas9-VP64 fusion protein and the MS2-P65-HSF1 coactivator.

[0017] Optionally, after synergistic activation of the expression of non-coding RNAs ENH323047, ENH264069 and ENSMUSG00000106683, the proportion of two-cell-like cells in mouse embryonic stem cells reached as high as 11.6%.

[0018] Thirdly, the present invention provides the application of the above-mentioned non-coding RNA combination in the preparation of cell products for regenerative medicine or in the construction of in vitro models for studying early embryonic development.

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

[0020] (1) This invention is the first to identify a functional combination consisting of specific enhancer RNAs (ENH323047 and ENH264069) and long non-coding RNAs (ENSMUSG00000106683), filling the gap in the understanding of the synergistic regulation of stem cell pluripotency by the "non-coding RNA combination", and providing a new key target module for understanding the epigenetic network of early development;

[0021] (2) This invention utilizes CRISPR activation (CRISPRa) technology to synergistically upregulate the expression of endogenous non-coding RNA combinations, thereby avoiding permanent integration of exogenous transcription factor genes and significantly reducing the genomic insertion mutations and potential safety risks associated with traditional overexpression methods.

[0022] (3) This invention achieves specific activation of endogenous genes through precise targeting mediated by guide RNA (sgRNA), overcoming the problems of widespread off-target effects across the entire genome and uncontrollable perturbation of cell state caused by chemical small molecule treatment, and has high manipulation specificity and reproducibility.

[0023] (4) This invention, by synergistically activating the combination of non-coding RNAs, has for the first time verified the synergistic effect of the combination in inducing pluripotency. Its efficiency in inducing mouse embryonic stem cells to transform into two-cell-like cells (2CLC) (the proportion of 2CLC is as high as about 11.6%) is far greater than that of activating a single non-coding RNA or a known transcription factor (such as Dux), providing a new strategy for efficient pluripotency reprogramming.

[0024] (5) The technical solution constructed by this invention has a high degree of standardization. Based on the mature CRISPRa vector system, the process is clear and highly reproducible, providing a stable and reliable new tool for basic stem cell research and regenerative medicine application development. It also lays an important technical foundation for cell fate modification based on endogenous gene network regulation, construction of early development in vitro models that are closer to physiological state, and development of safer regenerative medicine cell products. Attached Figure Description

[0025] Figure 1 The images show the expression profiles of three key non-coding RNAs (ENH323047, ENH264069, ENSMUSG00000106683) in various stages of early mouse embryonic development and in two-cell-like cells (2CLC). Specifically, (a) shows the expression profile of non-coding RNA ENH323047 in various stages of early mouse embryonic development and in two-cell-like cells (2CLC); (b) shows the expression profile of non-coding RNA ENH264069 in various stages of early mouse embryonic development and in two-cell-like cells (2CLC); and (c) shows the expression profile of non-coding RNA ENSMUSG00000106683 in various stages of early mouse embryonic development and in two-cell-like cells (2CLC).

[0026] Figure 2 The diagram shows the functional validation of the CRISPRa screening system constructed in this invention; (a) shows the gene expression levels of the negative control group (NC group) and the positive control group (Dux genome activated); (b) shows the expression levels of the totipotency marker gene after Dux gene activation in the positive control group; (c) shows the flow cytometry diagram of the negative control group (NC group); and (d) shows the flow cytometry diagram after Dux gene activation.

[0027] Figure 3The graphs show the expression levels of the target non-coding RNAs (ENH323047, ENH264069, ENSMUSG00000106683) and 2C marker genes (Mervl Pol, Zscan4c, Dux) after activation of the three key non-coding RNAs. Specifically, (a) shows the gene expression level after activation of non-coding RNA ENH323047; (b) shows the expression level of the 2C marker genes (Mervl Pol, Zscan4c, Dux) after activation of non-coding RNA ENH323047; (c) shows the gene expression level after activation of non-coding RNA ENH264069; (d) shows the expression level of the 2C marker genes (Mervl Pol, Zscan4c, Dux) after activation of non-coding RNA ENH264069; (e) shows the gene expression level after activation of non-coding RNA ENSMUSG00000106683; and (f) shows the expression level after activation of non-coding RNA... Expression level of 2C marker genes (Mervl Pol, Zscan4c, Dux) after ENSMUSG00000106683;

[0028] Figure 4 Flow cytometry plots after activation of three key non-coding RNAs are shown below; (a) is the flow cytometry plot of the negative control group (NC group); (b) is the flow cytometry plot after activation of non-coding RNA ENH323047; (c) is the flow cytometry plot after activation of non-coding RNA ENH264069; and (d) is the flow cytometry plot after activation of non-coding RNA ENSMUSG00000106683.

[0029] Figure 5 This is a verification diagram of the high efficiency of the present invention in inducing mouse embryonic stem cell pluripotency by synergistic activation of three key non-coding RNAs; where (a) and (b) are the percentages of 2C::tdTomato positive cells in the negative control group and the synergistic activation group under a fluorescence microscope, respectively; (c) and (d) are the percentages of 2C::tdTomato positive cells in the negative control group and the synergistic activation group (simultaneously activating ENH323047, ENH264069, and ENSMUSG00000106683), respectively.

[0030] Figure 6The graph shows the expression levels of three target non-coding RNAs (ENH323047, ENH264069, ENSMUSG00000106683) and three 2C marker genes (Mervl Pol, Zscan4c, Dux) in the co-activation group; where (a) is the gene expression level graph of the three target non-coding RNAs in the co-activation group; and (b) is the expression level graph of the 2C marker genes (Mervl Pol, Zscan4c, Dux) after the three target non-coding RNAs in the co-activation group. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical or scientific terms used herein should have the ordinary meaning understood by those skilled in the art to which this invention pertains.

[0032] This invention provides a combination of non-coding RNAs for inducing pluripotency in mouse embryonic stem cells, comprising: an enhancer RNA with the sequence shown in SEQ ID NO:1, an enhancer RNA with the sequence shown in SEQ ID NO:2, and a long non-coding RNA with the sequence shown in SEQ ID NO:3. In fact, this invention is the first to reveal how utilizing the CRISPR activation system to synergistically upregulate the expression of this specific combination of endogenous non-coding RNAs can efficiently promote the transformation of mouse embryonic stem cells into two-cell-like cells. This invention eliminates the need for exogenous transcription factor integration, avoiding genomic safety risks; it achieves precise targeting through sgRNA, overcoming the non-specific off-target defects of small chemical molecules; and it produces a significant synergistic effect, significantly increasing the induction efficiency of two-cell-like cells to approximately 11.6%. This invention provides a safer, more efficient, and controllable new strategy for stem cell fate reprogramming and early developmental research.

[0033] The present invention also provides an application of the above-mentioned non-coding RNA combination in inducing mouse embryonic stem cells to transform into a pluripotent two-cell-like cell state, which is achieved by synergistically upregulating the expression levels of three RNAs in the above-mentioned non-coding RNA combination.

[0034] In some embodiments, the above application is achieved by employing a CRISPR activation system to endogenously and synergistically activate the expression of three RNAs in a combination of non-coding RNAs.

[0035] In some embodiments, the selected CRISPR activation system includes guide RNAs that target the respective RNA regulatory regions in the combination of non-coding RNAs, the guide RNAs including:

[0036] (1) sgRNAs1 targeting the sequence regulatory region shown in SEQ ID NO:1, including SEQ ID NO:4 to SEQ ID NO:12;

[0037] (2) sgRNAs2 targeting the sequence regulatory region shown in SEQ ID NO:2, including SEQ ID NO:13 to SEQ ID NO:25;

[0038] (3) sgRNAs3 targeting the regulatory region of the sequence shown in SEQ ID NO:3, including SEQ ID NO:26 to SEQ ID NO:31.

[0039] In some embodiments, the CRISPR activation system used also includes the dCas9-VP64 fusion protein and the MS2-P65-HSF1 coactivator.

[0040] In some embodiments, after synergistic activation of the expression of non-coding RNAs ENH323047, ENH264069 and ENSMUSG00000106683, the proportion of two-cell-like cells in mouse embryonic stem cells was as high as 11.6%.

[0041] The present invention also provides an application of the above-mentioned non-coding RNA combination in the preparation of cell products for regenerative medicine or in the construction of in vitro models for studying early embryonic development. Example 1

[0042] Example 1 provides the identification of three key non-coding RNA targets.

[0043] Candidate targets were screened through bioinformatics analysis: Based on RNA-seq data of various stages of early mouse embryonic development published in public databases, and combined with non-coding RNA annotation, the expression profile of non-coding RNA was systematically analyzed.

[0044] The screening criteria were: 1) expression levels were significantly higher in two-cell stage embryos and 2CLC than in other stages and pluripotent embryonic stem cells (mESC); 2) high connectivity of non-coding RNA-mRNA association networks; and 3) predicted interaction with key regulatory proteins of totipotency fate transition.

[0045] Data analysis results: Three candidate non-coding RNAs were identified: ENH323047 (eRNA, SEQ ID NO:1), ENH264069 (eRNA, SEQ ID NO:2), and ENSMUSG00000106683 (lncRNA, SEQ ID NO:3). Visualization using IGV software confirmed that all three exhibit unique spatiotemporal expression patterns during mouse embryonic development.

[0046] like Figure 1 As shown, the transcriptional signals of these three molecules exhibit significant and specific high expression in pluripotent two-cell (2C) embryos and in vitro induced two-cell-like cells (2CLC). However, in earlier zygote stages or later pluripotency stages (such as blastocysts and embryonic stem cells), their expression levels remain extremely low or undetectable. These results strongly support, from a bioinformatics perspective, the rationale for ENH323047, ENH264069, and ENSMUSG00000106683 as key candidate targets for regulating pluripotency acquisition. Example 2

[0047] Example 2 provides the design of sgRNA sequences and plasmid construction.

[0048] sgRNA Design: Candidate sgRNAs were designed using the online tool CRISPOR (http: / / crispor.org) targeting the genomic regulatory regions (promoter / enhancer regions) of ENH323047, ENH264069, and ENSMUSG00000106683. The design principle prioritized sequences with high on-target efficiency prediction scores and low potential off-target effects. Specific targeting sgRNA sequence information is shown in SEQ ID NO:4 to SEQ ID NO:37. Simultaneously, as experimental controls, the positive control sgRNA targeted the regulatory region of the known 2C stage key activator, the Dux gene, while the negative control sgRNA (NC group) used sgRNAs targeting genomically irrelevant sites. The sequence information of all sgRNAs is shown in Table 1.

[0049] Table 1: sgRNA Sequence Information

[0050]

[0051]

[0052]

[0053] Plasmid construction:

[0054] sgRNA sequence annealing ligation: The configuration of the sgRNA sequence annealing ligation system is shown in Table 2 below.

[0055] Table 2: Configuration of sgRNA sequence annealing and ligation system

[0056]

[0057] After preparing the reaction system, place it in a constant temperature digital display water bath that has been preheated to 95°C and react for 5 minutes. Then turn off the heating switch of the constant temperature digital display water bath and wait for the temperature to drop naturally to room temperature to end the reaction.

[0058] Linearization of the sgRNA expression vector: This invention employs the CRISPRa system based on a three-plasmid system. To clone the synthesized sgRNA sequence into the sgRNA expression vector, the vector must first be linearized by enzyme digestion to provide the correct cloning site. In a PCR reaction tube, 5 μg of the sgRNA expression vector (Addgene #73797 or its equivalent), 3 μL of Esp3I (also known as BsmBI) restriction endonuclease, and 5 μL of the accompanying rCutSmart Buffer (NEB's CutSmart Buffer) are added sequentially. Finally, nuclease-free water is added to bring the reaction volume to 50 μL. The reaction is then performed at 37°C using a gene amplification instrument for 12 hours (or overnight) to ensure complete digestion. After the digestion reaction, the reaction solution is immediately purified using a commercial DNA fragment purification kit (Omega Bio-Tek Gel Extraction Kit D250) to recover the linearized vector fragment. The completeness of the digestion is verified by 1% agarose gel electrophoresis, and the concentration and purity of the recovered product are determined using a micro-spectrophotometer. The purified linearized vector was stored at -20°C for later use.

[0059] Finally, the purified linear vector and the annealed ligated sgRNA fragment were ligated using T4 ligase (T4 DNA Ligase from Dalian Takara Bio Co., Ltd.) to form the sgRNA expression plasmid. The enzyme ligation reaction system is shown in Table 3 below. The gene amplification instrument was set at 16℃ and the reaction was carried out for 12 hours.

[0060] Table 3: Enzyme Ligation Reaction System Configuration Table

[0061]

[0062] After the ligation reaction, 2-5 μL of the ligation product was mixed with competent cells (TOP10 from Sangon Biotech Co., Ltd.) and transformed using the standard heat shock method. The transformed bacterial culture was spread onto LB agar plates containing the appropriate antibiotics and incubated upside down in a 37°C incubator for 12-16 hours. 8-12 single colonies were randomly selected from the plates for PCR verification. The verification system is shown in Table 4 below (PCR enzyme was 2×Rapid Taq Master Mix from Nanjing Novizan Biotechnology Co., Ltd.). The reaction was performed using a gene amplification instrument with the following program: 95°C for 3 minutes; 95°C for 15 seconds, 55°C for 15 seconds, 72°C for 15 seconds, 32 cycles; 72°C for 5 minutes. After the reaction, the PCR products were detected by 1.5% agarose gel electrophoresis. Colonies showing the expected band size were preliminarily identified as positive clones.

[0063] Table 4: PCR Validation System Configuration Table

[0064]

[0065] Select 3-5 PCR-positive monoclonal colonies and inoculate them into LB broth containing antibiotics, incubating at 37°C and 220 rpm for 12-16 hours with shaking. Send an appropriate amount of the bacterial culture to a commercial sequencing service company for Sanger sequencing using the universal sequencing primers on the vector. Align the sequencing results with the designed sgRNA target sequence; clones with completely correct sequences are considered successfully constructed sgRNA expression plasmids. For the positive clones verified by sequencing, extract the plasmid using a commercial plasmid miniprep kit and determine its concentration and purity. Store the final plasmid at -20°C for subsequent cell experiments.

[0066] Example 3

[0067] Example 3 provides lentiviral packaging carrying each component of the CRISPRa system. Lentiviral viruses carrying the following three functional elements are packaged separately for subsequent construction of stable cell lines: dCas9-VP64 expression element (based on Addgene #61425, carrying blast fungicide resistance), MS2-P65-HSF1 expression element (based on Addgene #89308, carrying hygromycin resistance), and sgRNA expression element targeting three key non-coding RNAs (constructed based on Addgene #73797, carrying puromycin resistance).

[0068] 293T cell plating: Resuscitate and routinely culture 293T cells. Once the cells are stable and have grown to the 2nd or 3rd generation, digest them when the cell density reaches 90% and then plate them at an appropriate cell concentration.

[0069] Plasmid co-transfection: Transfection is performed when cells have adhered to the culture medium for approximately 12 hours and the confluence reaches 70%-80%. Before transfection, replace the cell culture medium with 9 mL of fresh, preheated complete medium (DMEM + 10% FBS + 1% penicillin-streptomycin). Prepare a new sterile EP tube and add the corresponding plasmids in the following ratios: packaging plasmid (PSPAX2): envelope plasmid (PMD2.G): target plasmid = 3:2:5. Mix slowly, with the target plasmid generally added at 7.5 μg, resulting in a total DNA content of 15 μg. Add 60 μL of PEI reagent at a ratio of PEI:DNA = 4:1 (v:m), mix gently, and let stand for 5 minutes. Then, add Opti-MEM solution to bring the mixture to 1 mL, mix well, and let stand for 15 minutes to form a stable DNA-transfection reagent complex. The complex solution was slowly added dropwise to the 293T cells, and the culture dish was gently shaken in a cross shape before being returned to the incubator for further culture.

[0070] Medium change: About 12 hours after transfection, replace the 293T cells with 10 mL of fresh culture medium and continue culturing.

[0071] Lentiviral fluid collection: Cell supernatant containing viral particles was collected into sterile centrifuge tubes at 48 hours and 72 hours after transfection.

[0072] Filtration: Centrifuge at 4℃ and 2000r for 10 minutes to initially clarify the virus stock solution and remove cell debris; then filter the supernatant using a 0.45 μm pore size low protein binding sterile filter membrane to further remove residual impurities.

[0073] Aliquoting and Storage: Aliquot the filtered virus stock solution into sterile cryovials according to the single-use quantity, and clearly label with virus information. Immediately store in an ultra-low temperature freezer at -80°C. To avoid a decrease in virus titer, strictly avoid repeated freeze-thaw cycles.

[0074] Example 4

[0075] Example 4 provides the construction and screening of stable cell lines using the CRISPRa system. Through stepwise infection and screening, mouse embryonic stem cell (mESC) lines that stably express each component of the CRISPRa system are constructed.

[0076] Preparation and plating of mESCs: One day in advance, add 1.5 mL of 0.1% gelatin to a six-well plate for plating. The next day, revive the cells and culture them in complete medium (2i medium) as usual. When the cells are stable and have grown to the 2nd or 3rd generation, digest them when the cell density reaches 90% and then plate them at an appropriate cell concentration.

[0077] Lentiviral infection: After cell plating, culture for approximately 12 hours until complete cell adhesion and a cell density of approximately 65%, discard the old culture medium. Mix the lentivirus stock solution carrying the dCas9-VP64 element, prepared according to Example 3, with fresh mESC complete culture medium at a 1:1 volume ratio, and add polybrene to a final concentration of 8 μg / mL. Gently add the mixture (2 mL total volume per well) to the wells, gently shake to mix, and incubate the plate at 37°C in a 5% CO2 incubator for 24 hours.

[0078] Infection and medium change: 24 hours after infection, carefully aspirate the virus-containing culture medium and replace it with fresh mESC complete culture medium. Continue culturing for 48-72 hours to allow the resistance gene to be fully expressed.

[0079] Antibiotic selection: Infected cells were passaged at an appropriate density into new gelatin-coated plates. After culturing for 12-16 hours, the medium was replaced with mESC selection medium containing an appropriate concentration of blastomycin (10 μg / mL). The antibiotic selection status of the cells was observed every 24 hours. During this period, the cells were regularly passaged and replaced with complete medium containing an appropriate concentration of antibiotic. After several days of culture (generally 4-7 days for antibiotic selection), 100% of the control group (uninfected cells) died, while the infected group still contained normally growing adherent cells. The surviving positive cell clones were expanded to obtain a stable mESC cell line that integrates and expresses the dCas9-VP64 element.

[0080] Based on the mESC cell line expressing the dCas9-VP64 element, the lentivirus stock solution carrying the MS2-P65-HSF1 expression element prepared in advance according to Example 3 was infected according to the same lentivirus infection procedure, and the cell lines expressing the MS2-P65-HSF1 element were screened for antibiotics for 4-7 days using mESC selection medium containing hygromycin (150 μg / mL).

[0081] Finally, based on the mESCs that stably express dCas9-VP64 and MS2-P65-HSF1 elements, the lentiviral stock solution carrying sgRNA expression elements prepared in advance according to Example 3 was infected according to the same lentiviral infection procedure, and antibiotic screening was performed for 4-7 days using mESC selection medium containing puromycin (1-2 μg / mL) to obtain positive cell lines that stably integrate and express all three CRISPRa components.

[0082] Example 5

[0083] Example 5 provides the verification of the activation and totipotency induction effects of non-coding RNAs using RT-qPCR and flow cytometry, aiming to verify: the specific activation efficiency of the CRISPRa system for three key non-coding RNAs; the promoting effect of this activation on the expression of totipotency marker genes; and the resulting change in the proportion of two-cell-like cells (2CLC).

[0084] Cellular RNA extraction and cDNA synthesis: Total RNA was extracted from cells in both experimental and control groups using a commercially available total RNA extraction kit (such as the SteadyPure Rapid RNA Extraction Kit from Acrel Biotech Co., Ltd.) according to the manufacturer's instructions. RNA concentration and purity (A260 / A280) were determined using a micro-spectrophotometer.

[0085] Take an equal volume (e.g., 1 μg) of total RNA and use a reverse transcription kit (e.g., HiScript III RT SuperMix for qPCR (+gDNA wiper) from Nanjing Novizan Biotechnology Co., Ltd.) to remove genomic DNA and synthesize cDNA. Follow the kit instructions for the reaction system and procedure. Dilute the obtained cDNA with nuclease-free water and store at -20°C for later use.

[0086] Real-time quantitative PCR (RT-qPCR) analysis: RT-qPCR was performed using the SYBR Green assay. The reaction mixture (20 μL) contained: 10 μL of 2×SYBR Green qPCR Master Mix, 0.8 μL each of forward and reverse primers (10 μM), 4 μL of cDNA template, and nuclease-free water to make up the volume. The specific primer sequences used are shown in Table 5.

[0087] Table 5: Primers for Real-Time Quantitative PCR

[0088]

[0089]

[0090] Flow cytometry analysis using transient transfection of reporter plasmid: This invention employs transient transfection of the 2C::tdTomato plasmid, and analyzes the fluorescence ratio of mcherry by flow cytometry to confirm the proportion of pluripotent stem cells. The 2C::tdTomato plasmid can be used to indicate the state of two-cell-like cells. Specific implementation steps are as follows:

[0091] The stable cell lines constructed in Example 4 were cultured in 6-well plates until confluence reached approximately 50%. Using a liposome transfection reagent (such as Lipofectamine 3000 from Thermo Fisher Scientific), 1.5 μg of the 2C::tdTomato reporter plasmid was transfected. Forty-eight hours post-transfection, the cells were digested to prepare single-cell suspensions, which were then analyzed using flow cytometry.

[0092] To verify the effectiveness of the constructed CRISPRa system, the Dux gene, a known key regulator of totipotency, was first used as a positive control. Figure 2 As shown, after activating Dux in mESCs, RT-qPCR analysis revealed that not only was Dux's expression efficiently upregulated, but the transcriptional levels of its classic 2C marker genes (such as Mervl Pol and Zscan4c) also significantly increased. Simultaneously, flow cytometry analysis showed that the proportion of 2C::tdTomato reporter positive cells increased more than 10-fold compared to the control group (negative control group). This confirms that the system can effectively upregulate Dux expression and subsequently promote the transformation of ESCs to the 2CLC pluripotent state. Therefore, the CRISPRa system used in this invention can effectively perform gene activation and successfully drive cells to the 2CLC state, providing a reliable technical platform for subsequent target screening.

[0093] Based on the validation of the system's effectiveness, further functional validation of single-target activation was performed on the three candidate non-coding RNAs. The results are as follows: Figure 3 As shown, when any single target site (ENH323047, ENH264069, or ENSMUSG00000106683) was activated individually, the expression of its corresponding non-coding RNA was specifically and significantly upregulated (p < 0.01). Notably, 2C marker genes (Mervl Pol, Zscan4c, Dux) also showed varying degrees of upregulation, indicating that each non-coding RNA possesses a certain totipotency function on its own.

[0094] Not only that, Figure 4 Cell loss analysis showed that single-target activation also led to an observable increase in the proportion of 2C::tdTomato positive cells. However, the proportion of 2CLC induced by each single-target activation group was much lower than that of the positive control (Dux activation group). This preliminarily confirms that these three non-coding RNAs are all pluripotent positive regulators with independent activity, but their individual effects are limited, suggesting the possibility of synergistic effects.

[0095] Based on the limited effectiveness but efficiency of single-target activation, the core scheme of this invention is implemented: synergistic activation of three non-coding RNAs. Its highly efficient induction effect is as follows: Figure 5 As shown: Fluorescence microscopy observation as follows Figure 5 Images (a) and (b) visually demonstrate that a large number of bright tdTomato positive cell clones appeared in the co-activation group, while almost none were seen in the negative control group (NC group). Figure 5 Quantitative analysis by flow cytometry in (c) and (d) further confirmed that the proportion of 2CLCs in the co-activation group reached 11.6%, which was not only significantly higher than that in the negative control group (p < 0.001), but also far exceeded that in any single-target activation group. Figure 4 (and the positive control Dux activation group)

[0096] Based on this, further RT-qPCR detection was performed, and the results were as follows: Figure 6 As shown, upon co-activation, the expression of non-coding RNAs at the three target sites was synchronously and efficiently upregulated. Simultaneously, all detected 2C marker genes (Mervl Pol, Zscan4c, Dux) exhibited co-regulated expression increases, with upregulation folds higher than those observed with single-target activation.

[0097] In summary, the specific non-coding RNA combination (ENH323047, ENH264069, ENSMUSG00000106683) provided by this invention can produce a strong synergistic effect under the synergistic activation of the CRISPRa system, thereby reprogramming mouse embryonic stem cells into two-cell-like cells with pluripotency characteristics with extremely high efficiency.

[0098] While embodiments of the present invention have been described in detail above, it will be apparent to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations fall within the scope and spirit of the invention as set forth in the claims. Furthermore, the invention described herein may have other embodiments and can be implemented or carried out in various ways.

Claims

1. A combination of non-coding RNAs for inducing pluripotency in mouse embryonic stem cells, characterized in that, The non-coding RNA combination includes: (1) The enhancer RNA with the sequence shown in SEQ ID NO:1 is named ENH323047; (2) The enhancer RNA with the sequence shown in SEQ ID NO:2 is named ENH264069; (3) The long non-coding RNA with the sequence shown in SEQ ID NO:3 is named ENSMUSG00000106683.

2. The application of the non-coding RNA combination as described in claim 1 in inducing the transformation of mouse embryonic stem cells into a pluripotent two-cell-like cell state, characterized in that, The application is achieved by synergistically upregulating the expression levels of three RNAs in the non-coding RNA combination.

3. The application according to claim 2, characterized in that, The application is achieved by using a CRISPR activation system to endogenously and synergistically activate the expression of three RNAs in a combination of non-coding RNAs.

4. The application according to claim 3, characterized in that, The CRISPR activation system includes guide RNAs that target the various RNA regulatory regions within the non-coding RNA combination, the guide RNAs comprising: (1) sgRNAs1 targeting the sequence regulatory region shown in SEQ ID NO:1, including SEQ ID NO:4 to SEQ ID NO:12; (2) sgRNAs2 targeting the sequence regulatory region shown in SEQ ID NO:2, including SEQ ID NO:13 to SEQ ID NO:25; (3) sgRNAs3 targeting the regulatory region of the sequence shown in SEQ ID NO:3, including SEQ ID NO:26 to SEQ ID NO:

31.

5. The application according to claim 4, characterized in that, The CRISPR activation system also includes the dCas9-VP64 fusion protein and the MS2-P65-HSF1 coactivator.

6. The use of a non-coding RNA combination as described in claim 1 in the preparation of cell products for regenerative medicine or in the construction of in vitro models for studying early embryonic development.

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