Enhancer RNA molecule MZGAe1 and use thereof

Abstract

The application provides an enhancer RNA molecule MZGAe1 and application thereof, and relates to the technical field of biology.The application provides an enhancer RNA molecule MZGAe1, and the nucleotide sequence of the enhancer RNA molecule MZGAe1 is shown in SEQ ID NO:1; meanwhile, the application also provides a specific sgRNA sequence for targeting and activating the molecule and a recombinant carrier thereof; by using the CRISPR activation technology, the expression of the endogenous MZGAe1 in cells can be specifically activated, and the mouse embryonic stem cells can be efficiently promoted to transform into two-cell-like cells. In the application, under the condition of endogenous activation of a single enhancer RNA molecule MZGAe1, the wild-type mouse embryonic stem cells can be efficiently transformed into two-cell-like cells, and the application provides a new efficient and specific research tool and scheme for studying zygote genome activation, early embryonic development events and cell reprogramming.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an enhancer RNA molecule, MZGAe1, and its applications. Background Technology

[0002] Zygote genome activation (ZGA) is the first major transcriptional event at the onset of mammalian life and plays a decisive role in whether an embryo can develop normally. The two-cell stage in mice is a critical window for the occurrence of major ZGA; therefore, two-cell cells are crucial for studying ZGA activation in developmental biology. Due to the extreme difficulty in obtaining in vivo embryonic material, researchers have discovered a rare, transient cell population spontaneously generated in mouse embryonic stem cells (mESCs)—two-cell-like cells (2CLCs)—whose transcriptome characteristics are highly similar to those of in vivo two-cell embryos, thus providing a vital in vitro model for studying ZGA and cell totipotency.

[0003] To utilize the 2CLC model, various schemes have been developed to increase its proportion, but many limitations remain: (1) Using chemical inducers (such as CX-5461, Aphidicolin) to treat mESCs can non-specifically induce 2CLC by causing transcriptional stress or DNA replication pressure. However, this chemical induction method has a wide range of effects and is accompanied by strong cytotoxicity and global transcriptional interference, resulting in a low signal-to-noise ratio in the experimental results; (2) Exogenous insertion of core transcription factors (such as DUX) can strongly initiate 2C-specific gene programs. Although this strategy of overexpressing core transcription factors is effective, it is too crude. Its non-physiological forced expression may submerge the endogenous fine regulatory network and may introduce artificial artifacts. Currently, there are few studies on the role of eRNA in the activation of the zygote genome in early embryos and the fate transition of mouse embryonic stem cells to two-cell-like cells. Moreover, there are very few studies on existing schemes for increasing the proportion of two-cell-like cells by endogenously activating bioactive RNA molecules.

[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 an enhancer RNA molecule MZGAe1 and its applications; by providing a novel enhancer RNA molecule MZGAe1 and a tool for precisely activating endogenous enhancer RNA molecules MZGAe1, this invention solves the technical problems of non-physiological forced transformation of two-cell-like cells in the prior art, which relies on non-specific chemical induction and lacks precise molecular targets.

[0006] In a first aspect, the present invention provides an enhancer RNA molecule MZGAe1, the nucleotide sequence of which is shown in SEQ ID NO:1.

[0007] Secondly, the present invention provides an sgRNA molecule that targets and activates the above-mentioned MZGAe1, wherein the targeting sequence of the sgRNA includes one of the sequences shown in SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0008] Thirdly, the present invention provides a recombinant vector comprising a nucleotide sequence encoding the aforementioned sgRNA molecule.

[0009] Optionally, the vector is a lentiviral vector.

[0010] Optionally, the vector's backbone includes an MS2 stem-loop sequence and a puromycin resistance selection marker.

[0011] Fourthly, the present invention provides the application of an enhancer RNA molecule, MZGAe1, including its use in increasing the proportion of two-cell-like cells in mouse embryonic stem cells.

[0012] Optionally, the application includes activating the expression of the pluripotency marker ZSCAN4 in the specific expression of the enhancer RNA molecule MZGAe1 in two-cell-like cells.

[0013] Fifthly, the present invention provides a method for increasing the proportion of two-cell-like cells in mouse embryonic stem cells, comprising: introducing a CRISPR gene activation system into the stem cells; the system being capable of specifically activating the expression of endogenous MZGAe1 in the stem cells.

[0014] Optionally, any of the above-mentioned optional recombinant vectors may be introduced into the stem cells to provide the CRISPR gene activation system.

[0015] Optionally, after activating the expression of MZGAe1, the proportion of the two-cell-like cells increases by at least 5%. Attached Figure Description

[0016] Figure 1 The bar chart shows the RNA expression levels in mouse embryonic stem cell control group and cells induced by the inducer CX-5461; where * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001, and t-test is used.

[0017] Figure 2The bar chart shows the RNA expression levels of mouse embryonic stem cell control group and MZGAe1 after endogenous activation using CRISPRa technology, as well as the RNA expression levels of two-cell phase-specific target genes after activation; where * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001, and t-test is used.

[0018] Figure 3 Flow cytometry analysis of the proportion of two-cell-like cells in mouse embryonic stem cell control group and mouse embryonic stem cells of MZGAe1 mice endogenously activated by CRISPR technology.

[0019] Figure 4 This is a schematic diagram of the recombinant vector containing the enhancer RNA molecule MZGAe1 sgRNA as a lenti sgRNA (MS2)_purooptimized backbone. Detailed Implementation

[0020] 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.

[0021] This invention provides an enhancer RNA molecule, MZGAe1, whose nucleotide sequence is shown in SEQ ID NO:1. Simultaneously, this invention also provides a specific sgRNA sequence for targeting and activating this molecule and its recombinant vector. By providing a novel endogenous tool for precisely activating MZGAe1, the transformation of mouse embryonic stem cells into two-cell-like cells is promoted. In fact, by specifically activating the expression of the endogenous enhancer RNA molecule MZGAe1 using CRISPR activation technology, the expression of the pluripotency marker gene ZSCAN4 can be precisely and specifically upregulated, efficiently promoting the transformation of mouse embryonic stem cells into two-cell-like cells, significantly increasing the proportion of two-cell-like cells, and obtaining a stable, high-quality two-cell-like cell model. This provides an efficient and specific new research tool and protocol for studying zygotic genome activation, early embryonic developmental events, and cell reprogramming.

[0022] This invention provides an enhancer RNA molecule, MZGAe1, whose nucleotide sequence is shown in SEQ ID NO:1. In fact, enhancers are a class of distal non-coding DNA regulatory elements that regulate cell type-specific gene expression through interaction with the promoters of their target genes; enhancers / superenhancers can be transcribed to produce enhancer RNA, which plays an important role in many biological processes. The enhancer RNA molecule MZGAe1 can be specifically expressed in two-cell-like cells and can activate the early embryonic developmental totipotency marker ZSCAN4, while also increasing the proportion of two-cell-like cells in mouse embryonic stem cells.

[0023] The present invention also provides an sgRNA molecule that targets and activates the enhancer RNA molecule MZGAe1, wherein the targeting sequence includes one of the sequences shown in SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0024] The present invention also provides a recombinant vector comprising the nucleotide sequence of an sgRNA molecule encoding the target activation enhancer RNA molecule MZGAe1.

[0025] In some embodiments, the recombinant vector is a lentiviral vector.

[0026] In some embodiments, the backbone of the recombinant vector includes the MS2 stem-loop sequence and a puromycin resistance selection marker.

[0027] The present invention also provides an application of the enhancer RNA molecule MZGAe1, including its use to increase the proportion of two-cell-like cells in mouse embryonic stem cells.

[0028] In some embodiments, the application of the enhancer RNA molecule MZGAe1 includes the activation of the expression of the pluripotency marker ZSCAN4 by MZGAe1 in specific expression in two-cell-like cells.

[0029] The present invention also provides a method for increasing the proportion of two-cell-like cells in mouse embryonic stem cells, comprising: introducing a CRISPR gene activation system into mouse embryonic stem cells; wherein the introduced CRISPR gene activation system is capable of specifically activating the expression of endogenous MZGAe1 in stem cells.

[0030] In fact, in the application of CRISPR gene activation (CRISPRa), a single-stranded RNA guide molecule (sgRNA) complementary to the genomic DNA site encoding the enhancer RNA molecule MZGAe1 was designed and introduced into mouse embryonic stem cells together with the Cas9 protein complex. The sgRNA guides Cas9 to locate the target DNA sequence and causes Cas9 to cut the DNA double strand at that location. When the cell repairs the DNA break caused by the cut, mutations can be introduced, thereby achieving gene knockout, insertion or replacement.

[0031] In some embodiments, the recombinant vector from any of the above embodiments is introduced into mouse embryonic stem cells to provide a CRISPR gene activation system.

[0032] In some embodiments, the proportion of two-cell-like cells increased by at least 5% after introducing the CRISPR gene activation system into mouse embryonic stem cells to activate the expression of MZGAe1.

[0033] It should be noted that the culture methods used in the following embodiments are commonly used in the prior art. The TRIzol method is a commonly used method for extracting RNA in the prior art. sgRNA design is a commonly used method for designing sgRNA in the prior art. The construction of enzyme digestion and ligation expression vectors is a commonly used method for vector construction in the prior art. Lentiviral packaging and infection is a commonly used method for constructing stable expression cell lines in the prior art. Flow cytometry is a cell analysis method in the prior art, which will not be described in detail here. Example

[0034] This embodiment 1 provides mouse embryonic stem cells that have not undergone any treatment and mouse embryonic stem cells induced by an inducer CX-5461, including the following steps:

[0035] First, mouse embryonic stem cells were cultured using the E14 cell line. The cells were placed in a 37°C, 5% CO2 cell culture incubator and cultured in a culture dish containing 1% gelatin and feeder cells. Once the mouse embryonic stem cells were cultured to a stable state, a control group and an experimental group were set up according to the experimental protocol. The control group received no treatment, while the experimental group was induced for 12 hours with 2 μM of the inducer CX-5461. Cells were then collected.

[0036] It should be noted that the use of an inducing agent to induce mouse embryonic stem cells to two-cell-like cells in Example 1 has been proven to be feasible. This induction method was used in Example 1 to preliminarily verify that MZGAe1 exists in two-cell-like cells and is specifically highly expressed. Example

[0037] Example 2 provides mouse embryonic stem cells containing an sgRNA containing the enhancer RNA molecule MZGAe1 and a lenti sgRNA (MS2)_puro optimized backbone recombinant vector containing a non-specific sgRNA that does not target any known gene or enhancer. The non-specific sgRNA serves as a negative control to ensure that the CRISPR system itself does not induce non-specific activation. The CRISPR system used in this laboratory has previously been shown to effectively activate target genes. Example 2 specifically includes the following steps:

[0038] S1. Culture of mouse embryonic stem cells; The mouse embryonic stem cell strain used was E14 cells, which were placed in a cell culture incubator at 37°C and 5% CO2 and cultured in a culture dish containing 1% gelatin and feeder cells.

[0039] Construction of S2 and sgRNA recombinant vectors:

[0040] (1) sgRNA design:

[0041] sgRNAs were designed using the CHOPCHOP online website. The sgRNA length is generally 20 nt. The principle for sgRNA sequence composition is that the gene-specific sgRNA template sequence precedes the PAM motif. The sgRNA sequence should avoid ending with more than four Ts, and the GC content is optimally 40%-60%. When constructing expression vectors driven by U6 or T7 promoters, the 5' base of the sgRNA should be G or GG to improve transcription efficiency. Table 1 shows the sgRNA sequences of the targeted activator enhancer RNA molecule MZGAe1 and non-specific sgRNAs not targeting any known genes or enhancers, obtained using the CHOPCHOP online website.

[0042] Table 1: sgRNA sequences targeting and activating MZGAe1, and non-specific sgRNA sequences.

[0043]

[0044] (2) Enzyme digestion and ligation:

[0045] Based on the experimental design, a suitable lentiviral vector was selected, digested with enzymes, and then gel-cleaved to obtain a linear vector. The designed and synthesized sgRNA was annealed and ligated overnight with the digested vector using T4 ligase. The ligation product was transformed into DH5α competent cells, and the plasmid was extracted. The successful construction of the vector was verified by enzyme digestion and colony PCR.

[0046] S3. Cell transfection and collection:

[0047] The cell transfection method used in this embodiment was lentiviral infection. The viral packaging system was a three-plasmid system, consisting of PSPA2, pMD2G, and a backbone vector. Commonly used virus packaging cells, 293T, were selected for lentiviral packaging. Viral fluid was collected at 48 and 72 hours post-transfection for concentration and purification. The concentrated and purified viral fluid was added dropwise to stable mouse embryonic stem cells. After 24 hours, cells were continuously screened with puromycin (1 μg / mL) for 7 days before collection. Examples 1 and 2 were both conducted at the Jiangxi Provincial Key Laboratory of Tumor Biology, Nanchang University.

[0048] Test Analysis:

[0049] The primers used in the real-time quantitative PCR analysis using RT-qPCR technology are shown in Table 2.

[0050] Table 2: Primers for Real-Time Quantitative PCR

[0051]

[0052] It should be noted that, as shown in Table 2, we designed a total of three pairs of RT-qPCR primers for MZGAe1. Figure 1 The results showed that all three primer pairs could specifically amplify MZGAe1. Figure 1 and Figure 2 The horizontal axis of the bar chart, MZGAe1-1, MZGAe1-2, and MZGAe1-3, represents the target gene MZGAe1 obtained after amplification using the three pairs of RT-qPCR primers (qRT-PCR-MZGAe1-F1 and qRT-PCR-MZGAe1-R1, qRT-PCR-MZGAe1-F2 and qRT-PCR-MZGAe1-R2, qRT-PCR-MZGAe1-F3 and qRT-PCR-MZGAe1-R3) listed in the table above.

[0053] RNA was extracted from mouse embryonic stem cells obtained in Example 1 using the TRIzol method, and the expression level of MZGAe1 was analyzed by RT-qPCR. Three independent biological replicates were performed, and the results are as follows: Figure 1 As shown.

[0054] Figure 1 The results showed that there was a significant difference in the expression of the enhancer RNA molecule MZGAe1 gene between the untreated control group and the CX-5461-induced experimental group, suggesting that the MZGAe1 gene exists in two-cell-like cells and is specifically highly expressed.

[0055] RNA was extracted from mouse embryonic stem cells obtained in Example 2 using the TRIzol method, and then the expression levels of MZGAe1, the pluripotency marker ZSCAN4, and other two-cell stage-specific expression genes (Duxf3, Mervl) were analyzed using RT-qPCR. The test results are as follows: Figure 2 As shown.

[0056] Figure 2 The results showed that there was a significant difference in the expression of the enhancer RNA molecule MZGAe1 gene between the non-specifically activated control group and the experimental group with endogenous MZGAe1 activation via CRISPR technology, indicating that the MZGAe1 gene was effectively activated. Furthermore, RT-qPCR analysis of the expression levels of the pluripotency marker ZSCAN4 and other two-cell phase-specific genes Duxf3 and Mervl in both the control and experimental groups with endogenous MZGAe1 activation via CRISPR technology revealed that, compared with the control group, the expression levels of the pluripotency marker ZSCAN4 and the two-cell phase-specific genes Duxf3 and Mervl were significantly upregulated in the experimental group, suggesting that some pluripotent mouse embryonic stem cells transformed into pluripotent two-cell-like cells.

[0057] Subsequently, the mouse embryonic stem cells obtained in Example 2 from the control and experimental groups were further cultured and transfected with the indicator fluorescent plasmid 2C::tdtomato. After 48 hours, cell suspensions were collected and analyzed by flow cytometry. Flow cytometry analysis can characterize the proportion of two-cell-like cells in mouse embryonic stem cells. The test results are as follows: Figure 3 As shown.

[0058] right Figure 3 The results analysis revealed that, compared with the control group, endogenous activation of the enhancer RNA molecule MZGAe1 increased the proportion of two-cell-like cells, which helps us to further study the role and molecular mechanism of the enhancer RNA molecule MZGAe1 in early mouse embryonic development.

[0059] 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. An enhancer RNA molecule MZGAe1 Its application in increasing the proportion of two-cell-like cells in mouse embryonic stem cells is characterized by... The application includes culturing mouse embryonic stem cells; introducing a CRISPR gene activation system into the mouse embryonic stem cells; the CRISPR gene activation system being able to specifically activate the endogenous genes of the mouse embryonic stem cells. MZGAe1 The expression of the CRISPR gene activation system is provided via a recombinant vector containing an sgRNA molecule; the sgRNA molecule targets and activates the gene. MZGAe1 The target sequence includes one of the sequences shown in SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4; MZGAe1 The nucleotide sequence is shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that, The recombinant vector is a lentiviral vector; the backbone of the recombinant vector includes the MS2 stem-loop sequence and a puromycin resistance selection marker.

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