Non-coding RNA combination and application thereof in mouse totipotent stem cell induction

Abstract

The application provides a non-coding RNA combination and application thereof in mouse totipotent stem cell induction, and relates to the technical field of biotechnology.The combination comprises: an enhancer RNA with a sequence as shown in SEQ ID NO:1 and SEQ ID NO:2, and a long-chain non-coding RNA with a sequence as shown in SEQ ID NO:3.The application discloses, for the first time, that the expression of the endogenous non-coding RNA combination is synergistically inhibited by using a CRISPR inhibition system, and the mouse embryonic stem cells can be efficiently promoted to transform into 2C-like cells.The application does not need to integrate an exogenous transcription factor, avoids the safety risk of the genome, overcomes the defect of non-specific off-target of a chemical small molecule by precise targeting, and significantly improves the induction efficiency of the 2C-like cells to about 9.09%.The application provides a safer, more efficient and controllable new strategy 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 and its application in the induction of mouse pluripotent stem cells. Background Technology

[0002] Mouse pluripotent stem cells (PPCs) are a class of cells capable of differentiating into embryonic tissues (all somatic cells) and extraembryonic tissues (placenta, yolk sac), and have broad application prospects in fields such as early embryonic development research, gene editing, disease model construction, and regenerative medicine. Currently reported mouse PPCs include zygotes, 2-cell embryonic (2C) stage cells, and 2C-like embryonic stem cells (2C-like ESCs). Among these, 2CLCs are currently a research hotspot due to their in vitro culture capability and pluripotency.

[0003] Currently, the existing technologies for inducing mouse pluripotent stem cells include the following: (1) Induction by compounds: For example, Wnt pathway activators (such as CHIR99021) can promote the transformation of cells to the pluripotent state, but there are problems such as low induction efficiency (usually less than 10%), strong cell heterogeneity, and unclear mechanism of action; (2) Achieved by overexpression of transcription factors: For example, pluripotent core transcription factors (such as Zscan4, Dppa3, etc.) are introduced exogenously to forcibly activate the pluripotent pathway, but there are disadvantages such as the risk of exogenous gene integration (some transcription factors may increase the risk of cell carcinogenesis) and high cost; (3) Single endogenous factor manipulation: In recent years, the role of endogenous non-coding RNA in cell fate regulation has been gradually recognized. However, the current application of non-coding RNA in the induction of mouse pluripotent stem cells is mostly limited to "single molecules", with limited regulatory effects, and there is a lack of systematic screening and verification of "non-coding RNA combinations", especially the systematic identification of eRNA and lncRNA molecular combinations. Due to its functional redundancy and the complexity of its mechanism of action, studying its 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 and their application in the induction of mouse pluripotent stem cells. 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 comprising:

[0007] (1) The enhancer RNA with the sequence shown in SEQ ID NO:1 is named ENH095415;

[0008] (2) The enhancer RNA with the sequence shown in SEQ ID NO:2 is named ENH151862;

[0009] (3) The long non-coding RNA with the sequence shown in SEQ ID NO:3 is named ENSMUSG00000099370.

[0010] Secondly, the present invention provides the application of the above-mentioned non-coding RNA combination in inducing the transformation of mouse pluripotent stem cells into 2C-like cells.

[0011] Optionally, the application includes inducing the transformation of mouse pluripotent stem cells into 2C-like cells by inhibiting the expression of the non-coding RNA combination.

[0012] Optionally, the expression of a combination of non-coding RNAs is suppressed using a CRISPR interference system; the CRISPR interference system comprises sgRNAs that target each RNA in the combination of non-coding RNAs; the sgRNAs include:

[0013] (1) sgRNA1 targeting the sequence regulatory region shown in SEQ ID NO:1, including one of SEQ ID NO:4 to SEQ ID NO:19;

[0014] (2) sgRNA2 targeting the sequence regulatory region shown in SEQ ID NO:2, including one of SEQ ID NO:20 to SEQ ID NO:35;

[0015] (3) sgRNA3 targeting the regulatory region of the sequence shown in SEQ ID NO:3, including one of SEQ ID NO:36 to SEQ ID NO:41.

[0016] Optionally, the CRISPR interference system further comprises a fusion protein of an inactivated Cas9 protein and a transcriptional repressor domain.

[0017] Optionally, after synergistic inhibition of the expression of non-coding RNAs ENH095415, ENH151862 and ENSMUSG00000099370, the proportion of 2C-like cells in mouse embryonic stem cells was as high as 9.09%.

[0018] Thirdly, the present invention 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.

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

[0020] (1) The non-coding RNA combination (ENH095415, ENH151862, ENSMUSG00000099370) and application method provided by the present invention, by jointly inhibiting the expression of the three, produced a significant synergistic regulatory effect, which increased the induction efficiency of mouse embryonic stem cells into 2C-like cells to about 9.09%, which is much higher than the effect of inhibiting a single molecule.

[0021] (2) The present invention uses CRISPR interference (CRISPRi) technology to inhibit target expression. This technology does not cut genomic DNA and achieves reversible and specific gene silencing, thereby avoiding the safety risks and potential carcinogenicity caused by exogenous gene integration in traditional overexpression methods.

[0022] (3) Compared with traditional small molecule compound induction, the mechanism of action of this invention is more explicit, directly targeting key nodes of the endogenous regulatory network, and inducing a more stable and uniform cell state;

[0023] (4) This invention innovatively adopts a “reverse regulation” strategy, that is, by inhibiting the non-coding RNA that is highly expressed in the target state (2C-like cells), the cell fate is reversed from pluripotency to totipotency, providing a new technical path for cell fate regulation;

[0024] (5) The non-coding RNA combination identified in this invention provides a novel key target and regulatory network module for studying the molecular mechanism of the transition from pluripotency to totipotency in early mouse embryonic development; and the corresponding sgRNA combination and corresponding vector tools provide a ready-to-use, standardized technical solution and product basis for the efficient and safe induction of mouse totipotent stem cells, which has good application prospects. Attached Figure Description

[0025] Figure 1 These are IGV visualizations of non-coding RNAs ENH095415, ENH151862, and ENSMUSG00000099370 as described in this invention; wherein, (a) is an IGV visualization of non-coding RNA ENH095415; (b) is an IGV visualization of non-coding RNA ENH151862; and (c) is an IGV visualization of non-coding RNA ENSMUSG00000099370.

[0026] Figure 2The following are RT-qPCR diagrams used in this invention to detect the inhibitory effects of the positive control Lin28 and non-coding RNAs (ENH095415, ENH151862, and ENSMUSG00000099370) in mouse embryonic stem cells; wherein, (a) is an RT-qPCR diagram to detect the inhibitory effect of the positive control Lin28 in mouse embryonic stem cells; (b) is an RT-qPCR diagram to detect the inhibitory effect of the non-coding RNA ENH095415 in mouse embryonic stem cells; (c) is an RT-qPCR diagram to detect the inhibitory effect of the non-coding RNA ENH151862 in mouse embryonic stem cells; and (d) is an RT-qPCR diagram to detect the inhibitory effect of the non-coding RNA ENSMUSG00000099370 in mouse embryonic stem cells.

[0027] Figure 3 These are flow cytometry plots showing the effects of individual non-coding RNA groups in this invention; (a) is a flow cytometry plot showing the percentage of 2C-like cells in the control group (NC); (b) is a flow cytometry plot showing the percentage of 2C-like cells in the positive control group (Lin28); (c) is a flow cytometry plot showing the percentage of 2C-like cells in the ENH095415 group; (d) is a flow cytometry plot showing the percentage of 2C-like cells in the ENH151862 group; and (e) is a flow cytometry plot showing the percentage of 2C-like cells in the ENSMUSG00000099370 group.

[0028] Figure 4 This diagram shows the expression levels of 2C-related marker genes (Mervl Pol, Zscan4c, Dux) in mouse embryonic stem cells knocked down by the positive controls Lin28, ENH095415, ENH151862, and ENSMUSG00000099370, as detected by RT-qPCR in this invention (under the action of a single non-coding RNA group). Specifically, (a) shows the expression levels of 2C-related marker genes (Mervl Pol, Zscan4c, Dux) in mouse embryonic stem cells knocked down by the positive control Lin28, as detected by RT-qPCR; (b) shows the expression levels of 2C-related marker genes (Mervl Pol, Zscan4c, Dux) in mouse embryonic stem cells knocked down by ENH095415, as detected by RT-qPCR; and (c) shows the expression levels of 2C-related marker genes (Mervl Pol, Zscan4c, Dux) in mouse embryonic stem cells knocked down by ENH095415, as detected by RT-qPCR. (d) is a graph showing the expression levels of 2C-related marker genes (Mervl Pol, Zscan4c, Dux) in ENH151862 knockdown mouse embryonic stem cells; (d) is a graph showing the expression levels of 2C-related marker genes (Mervl Pol, Zscan4c, Dux) in ENSMUSG00000099370 knockdown mouse embryonic stem cells detected by RT-qPCR.

[0029] Figure 5 In this invention, the inhibitory effect of non-coding RNA combinations on mouse embryonic stem cells was detected by RT-qPCR (under the induction of non-coding RNA combinations). Figure 5 The expression levels of a) and 2C-related marker genes (Mervl Pol, Zscan4c, Dux) in mouse embryonic stem cells with non-coding RNA combinatorial knockdown are shown in the figure. Figure 5 (b)

[0030] Figure 6 The images show the proportion of mouse embryonic stem cells 2C-like cells detected by fluorescence microscopy and flow cytometry under the induction of non-coding RNA combinations in this invention. Among them, (a) is a microscopic photograph of the proportion of 2C-like cells in the control group (NC); (b) is a microscopic photograph of the proportion of 2C-like cells under the induction of non-coding RNA combinations; (c) is a flow cytometry image of the proportion of 2C-like cells in the control group (NC); and (d) is a flow cytometry image of the proportion of 2C-like cells under the induction of non-coding RNA combinations. 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 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. Specifically, this invention is the first to reveal how utilizing the CRISPR inhibition system to synergistically inhibit 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 2C-like cells to approximately 9.09%. This invention provides a safer, more efficient, and controllable new strategy for stem cell fate reprogramming and early developmental research.

[0033] This invention also provides the application of a combination of non-coding RNAs (ENH095415, ENH151862, ENSMUSG00000099370) in inducing the transformation of mouse pluripotent stem cells into 2C-like cells.

[0034] In some embodiments, the induction of mouse pluripotent stem cell conversion into 2C-like cells is achieved by inhibiting the expression of a combination of non-coding RNAs (ENH095415, ENH151862, ENSMUSG00000099370).

[0035] In some embodiments, the expression of a combination of non-coding RNAs (ENH095415, ENH151862, ENSMUSG00000099370) is suppressed using a CRISPR interference system; the CRISPR interference system used comprises sgRNAs that target each RNA in the non-coding RNA combination; the selected sgRNAs include:

[0036] (1) sgRNA1 targeting the sequence regulatory region shown in SEQ ID NO:1, including one of SEQ ID NO:4 to SEQ ID NO:19;

[0037] (2) sgRNA2 targeting the sequence regulatory region shown in SEQ ID NO:2, including one of SEQ ID NO:20 to SEQ ID NO:35;

[0038] (3) sgRNA3 targeting the regulatory region of the sequence shown in SEQ ID NO:3, including one of SEQ ID NO:36 to SEQ ID NO:41.

[0039] In some embodiments, the CRISPR interference system also includes a fusion protein of an inactivated Cas9 protein and a transcriptional repressor domain.

[0040] In some embodiments, the proportion of 2C-like cells in mouse embryonic stem cells was as high as 9.09% after synergistic inhibition of the expression of non-coding RNAs ENH095415, ENH151862 and ENSMUSG00000099370.

[0041] The present invention also provides the application of a combination of non-coding RNAs (ENH095415, ENH151862, ENSMUSG00000099370) in the preparation of cell products for regenerative medicine or in the construction of in vitro models for studying early embryonic development.

[0042] In this embodiment of the invention, the cell lines used were 2R-E4-KRAB cells (2R refers to 2C::tdTomato, ZSCAN4-EGFP dual fluorescent cells), Mef cells, and 293T cells, all of which were cultured in an incubator at 37°C containing 5% CO2. Example 1

[0043] Example 1 provides a screening of three key non-coding RNAs (ENH095415, ENH151862, ENSMUSG00000099370).

[0044] By downloading published datasets of total RNA sequencing and polyA-enriched RNA sequencing at different developmental stages of early mouse embryos (Hua Yu, Jing Zhao, Yuxuan Shen, et al. The dynamic landscape of enhancer-derived RNA during mouse early embryo development [J]. Cell Reports, 2024, 43(4)), and through relevant bioinformatics analysis, with the assistance of IGV software and the UCSC website, candidate non-coding RNAs that are highly expressed during the ICM stage of embryonic development and highly expressed during the ESC stage of cell development, and with a number of linked genes greater than or equal to 10, were finally selected as subsequent targets. After relevant validation, three non-coding RNAs, ENH095415, ENH151862 and ENSMUSG00000099370, were finally selected as subsequent targets. Example 2

[0045] Example 2 provides the design of the sgRNA gene sequence and the construction of the plasmid.

[0046] sgRNA sequence template synthesis: Specific sgRNAs targeting sequences ENH095415 and ENH151862 near the transcription start site and ENSMUSG00000099370 targeting sequences in the promoter region were designed. Eight sgRNAs were designed for each of ENH095415 and ENH151862, and three sgRNAs were designed for ENSMUSG00000099370. Three sgRNAs were used as negative control (irrelevant RNA), and three sgRNAs were used as positive control (Lin28). Oligo templates (25 nt in length) were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and their sequences are shown in SEQ ID No: 4 to SEQ ID No: 53. sgRNA design is a common technique in this field. Those skilled in the art can also choose to use sgRNA design software or perform online design, which will not be elaborated here. The sequence information of all sgRNAs is shown in Table 1.

[0047] Table 1: sgRNA Sequence Information

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054] Plasmid construction:

[0055] sgRNA sequence annealing: The T4 ligase buffer reagent from TAKALA's T4 DNA ligation kit was used. The sgRNA sequence annealing reaction system was prepared as shown in Table 2 below:

[0056] Table 2: Configuration of sgRNA sequence annealing reaction system

[0057]

[0058] After preparing the system, mix it thoroughly, incubate at 95℃ for 5 minutes in a water bath, turn off the heating and allow it to cool naturally to room temperature before storing at -20℃ for later use.

[0059] Vector digestion: The vector used was LentiGuide-puro. The vector was digested, and the digestion products were subjected to agarose gel electrophoresis to verify their integrity and then recovered from the gel (OmeGa, D2500 Gel Extraction Kit). The NEB Esp3 I restriction endonuclease reagent was used, and the digestion reaction system was prepared according to Table 3 below:

[0060] Table 3: Configuration Table of Enzyme Digestion Reaction System

[0061]

[0062] After preparing the system, mix it well. The enzyme digestion reaction procedure is: 37°C overnight, then store at -20°C for later use.

[0063] Enzyme ligation: The T4 DNA ligation reagent from TAKALA was used, and the reaction system was prepared according to Table 4 below:

[0064] Table 4: Enzyme Ligation Reaction System Configuration Table

[0065]

[0066] After preparing the system, mix it well. Enzyme ligation reaction procedure: 16℃ overnight, then store at -20℃ for later use.

[0067] Transformation: The enzyme ligation product was transformed to verify whether the ligation was successful for subsequent colony PCR. The transformation reagent used was Top10 competent cells from Sangon Biotech (Shanghai) Co., Ltd.

[0068] Colony PCR: The reagents used were the 2x rapid Taq premix solution from Nanjing Novizan Biotechnology Co., Ltd., and the reaction system was prepared according to Table 5 below:

[0069] Table 5: Configuration Table of Colony PCR Reaction System

[0070]

[0071] After preparing the reaction system, mix it thoroughly and perform colony PCR according to the reaction procedure described in Table 6 below:

[0072] Table 6: Data on Colony PCR Reaction Process

[0073]

[0074] After colony PCR reaction, agarose gel electrophoresis was performed. Then, the positive bacteria were sent for sequencing (by Youkang Biotechnology Co., Ltd.). The sequencing results were then compared with the SnapGene software. The sequences that matched correctly were the successfully constructed sgRNA plasmids. Plasmids were extracted from the positive clones that were confirmed by sequencing, and their concentration was measured. Finally, the plasmids were stored at -20℃ for later use. The plasmid extraction kit used was the plasmid mini-prep kit from TIANGEN.

[0075] Example 3: This Example 3 provides individual (single-molecule) functional verification of the effects of three non-coding RNAs, ENH095415, ENH151862 and ENSMUSG00000099370.

[0076] Sample collection: The designed sgRNAs were cloned into the LentiGuide-puro vector (addgene#52963). Eight sgRNAs designed by ENH095415 and ENH151862, three sgRNAs designed by ENSMUSG00000099370, an irrelevant control sgRNA (gRNA-NC) (control group), and three positive control sgRNAs (sgRNA-Lin28) (positive control group) were mixed in equal amounts and packaged into lentivirus. These were then used to infect 2R-E14-KRAB cells, which were cultured in normal medium for at least two days until they stabilized. After passage, puro was added the following day for selection. Cells surviving 3-6 days after selection were analyzed by flow cytometry and RT-qPCR.

[0077] The specific primer sequence information used in real-time quantitative PCR (RT-qPCR) analysis is shown in Table 7.

[0078] Table 7: Primers for Real-Time Quantitative PCR

[0079]

[0080]

[0081] In this embodiment of the invention, the flow cytometry analysis method is as follows: To detect the phenotypic characteristics of mouse embryonic stem cells, the cells used in this invention are dual-fluorescent ES cells stably expressing the KRAB repression domain. Cell processing: Wash cells twice with pre-chilled PBS, digest with trypsin, centrifuge at 1500 rpm for 5 min, resuspend and wash 1-2 times with pre-chilled PBS, centrifuge at 1100 rpm for 5 min, carefully aspirate the supernatant, resuspend in 250 μL of pre-chilled PBS, mix thoroughly, filter through a 70 μm mesh filter, and then perform cell detection using a flow cytometer (Sony ID7000, Shanghai Sony Imaging Co., Ltd., China).

[0082] In this embodiment of the invention, the RNA preparation and quantitative real-time PCR (RT-PCR) methods are as follows: Total RNA was isolated from cells using the SteadyPuro Rapid RNA Extraction Kit (AG, China) according to the manufacturer's instructions, and reverse transcription was performed using the HiScript IIIRT SuperMix for qPCR (+gDNA wiper) kit (Vazyme Biotech Co Ltd, China). Gene expression was then detected by qRT-PCR using the ChamQ Universal SYBR qPCR Master Mix kit (Vazyme Biotech Co Ltd, China). mRNA expression levels were measured using the CT method, and GAPDH expression was used as a standardized indicator.

[0083] The results of the assays to verify the induction effect of a single non-coding RNA group are as follows:

[0084] Inhibitory effect of CRISPRi cell line detected by RT-qPCR: Results are as follows Figure 2 As shown, compared with the control group NC, the positive control Lin28 and non-coding RNAs: ENH095415, ENH151862 and ENSMUSG00000099370 were effectively inhibited in mouse embryonic stem cells.

[0085] Flow cytometry analysis of the proportion of 2C-like cells (using 2C::tdTomato as a marker): Results are as follows Figure 3 As shown, inhibition of the Lin28 gene increased the proportion of tdTomato positive cells to 2.49%, inhibition of ESENH095415 increased the proportion to 3.03%, inhibition of ENH151862 increased the proportion to 5.71%, and inhibition of ENSMUSG00000099370 increased the proportion to 3.37%, while the control group had only 0.68%.

[0086] RT-qPCR was used to detect the expression of 2C-related marker genes (Mervl Pol, Zscan4c, Dux) in mouse embryonic stem cells knocked down by positive controls Lin28, ENH095415, ENH151862, and ENSMUSG00000099370: The results are as follows. Figure 4 As shown, Mervl Pol expression increased by approximately 2-6 times, Zscan4c expression increased by approximately 2-10 times, and Dux expression increased by approximately 2-4 times.

[0087] In the embodiments of the present invention, FlowJo_v10.8.1 software (FlowJo LLC, America) is used to analyze and utilize the flow cytometry results, and GraphPadPrism10.1.2 software (San Diego, CA, USA) is used to perform statistical analysis on the qPCR results.

[0088] Example 4: This example provides functional validation of the effects of three non-coding RNA combinations (combinations): ENH095415, ENH151862, and ENSMUSG00000099370. The sample collection, flow cytometry analysis, and qPCR detection procedures for the combination induction experiment are detailed in Example 3 and will not be repeated here.

[0089] The induction efficiency of the non-coding RNA combinations (ENH095415, ENH151862, and ENSMUSG00000099370) is as follows:

[0090] RT-qPCR was used to detect the inhibitory effect of a combination of non-coding RNAs (ENH095415, ENH151862, and ENSMUSG00000099370) on mouse embryonic stem cells. The results are as follows: Figure 5 and Figure 6 As shown, the inhibition effect is significant.

[0091] RT-qPCR detection of 2C-related marker genes (Mervl Pol, Zscan4c, Dux) yielded the following results: Figure 5 As shown, Mervl Pol expression was increased by about 7-fold, Zscan4c expression by about 14-fold, and Dux expression by about 9-fold in the combination group, which were significantly higher than those in the single-molecule group.

[0092] Flow cytometry analysis of the proportion of 2C-like cells (using 2C::tdTomato as a marker): Results are as follows Figure 6 As shown, the proportion of tdTomato positive cells in the combination group was 9.09%, while that in the control group was 0.68%; this represents an increase of approximately 1.5-3 times compared to the single-molecule combination.

[0093] In summary, this implementation scheme demonstrates that combining non-coding RNA can significantly improve the induction efficiency of mouse pluripotent stem cells, achieving higher induction efficiency than traditional induction methods, thereby solving the problem of "low induction efficiency".

[0094] 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 non-coding RNA combination, characterized in that, The non-coding RNA combination includes: (1) The enhancer RNA with the sequence shown in SEQ ID NO:1 is named ENH095415; (2) The enhancer RNA with the sequence shown in SEQ ID NO:2 is named ENH151862; (3) The long non-coding RNA with the sequence shown in SEQ ID NO:3 is named ENSMUSG00000099370.

2. The application of the non-coding RNA combination as described in claim 1 in inducing the transformation of mouse pluripotent stem cells into 2C-like cells, characterized in that, The application involves inducing the transformation of mouse pluripotent stem cells into 2C-like cells by inhibiting the expression of the non-coding RNA combination.

3. The application according to claim 2, characterized in that, The expression of a combination of non-coding RNAs is inhibited by a CRISPR interference system; the CRISPR interference system comprises sgRNAs that target each RNA in the combination of non-coding RNAs; the sgRNAs include: (1) sgRNA1 targeting the sequence regulatory region shown in SEQ ID NO:1, including one of SEQ ID NO:4 to SEQ ID NO:19; (2) sgRNA2 targeting the sequence regulatory region shown in SEQ ID NO:2, including one of SEQ ID NO:20 to SEQ ID NO:35; (3) sgRNA3 targeting the regulatory region of the sequence shown in SEQ ID NO:3, including one of SEQ ID NO:36 to SEQ ID NO:

41.

4. The application according to claim 3, characterized in that, The CRISPR interference system also includes a fusion protein of an inactivated Cas9 protein and a transcriptional repressor domain.

5. 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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