An epigenetic gene editing system based on CRISPR / dCas12a and its application

Abstract

The present invention relates to the field of genetic engineering technology, and discloses an epigenetic gene editing system based on CRISPR / dCas12a and its application. Wherein, the epigenetic gene editing system comprises: a superCRISPRon fusion protein and a crRNA that guides the superCRISPRon fusion protein to target a target gene; the amino acid sequence of the superCRISPRon fusion protein is shown in any one of SEQ ID NO.1~6. Compared with TET1-dCas12a and TET1-dCas12a-VPR, the epigenetic gene editing system of the present invention has the characteristics of high efficiency and lasting effect. In addition, the epigenetic gene editing system of the present invention can activate the expression of follistatin (FSTN) in muscle satellite cells, improve the differentiation ability of muscle satellite cells, and thus promote muscle growth. The epigenetic gene editing system upgrades the existing tools, improves the editing efficiency and duration, and plays an important role in promoting the application of gene editing in clinical treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to an epigenetic gene editing system based on CRISPR / dCas12a and applications thereof. Background Art

[0002] Epigenetic gene editing refers to changing the expression level of a gene by changing DNA methylation deposition or chromatin conformation without changing the DNA sequence. Combining dCas protein with epigenetic factors can recruit epigenetic proteins to target genes for specific gene activation or inhibition. Transcription factors VP64 or p65 are fused to dCas9 protein. These transcription activators can recruit transcription initiation complexes in the promoter region to activate genes at the transcriptional level, and multiple sgRNAs have a synergistic effect. The three transcription activation domains of VP64, p65 and Rta are fused in series to the dCas9 protein to form the dCas9-VPR system, which has a better endogenous target gene activation effect and a stable multi-locus activation effect. DNA demethylase TET1 (Ten-eleven translocation methylcytosinedioxygenase) can catalyze the conversion of 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) intermediates, mediating active DNA demethylation through oxidation and base excision repair (BER) pathways. Researchers fused TET1 with dCas9 protein to produce DNA demethylation in the promoter region of the target gene and induce gene re-expression. However, the efficiency and duration of current gene activation tools still need to be improved.

[0003] The CRISPR / Cas12a system is currently the main gene editing technology. The Cas12a protein is a smaller and simpler nuclease than the Cas9 protein, with only 1200~1300 amino acids. It can mediate strong DNA interference and has different physiological characteristics from the Cas9 protein. The Cas12a protein belongs to the type II type VA CRISPR system. Like the Cas9 protein, the Cas12a protein can bind to the target genomic site and produce double-strand breaks, which are then repaired by non-homologous end-joining (NHEJ) or homology directed repair (HDR). Compared with the Cas9 protein, the Cas12a protein has a lower off-target effect and has great potential in gene therapy applications. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide an epigenetic gene editing system based on CRISPR / dCas12a and its application, aiming to solve the problems of low transcriptional activation efficiency and short activation maintenance time of the existing epigenetic gene editing system.

[0005] The technical solution of the present invention is as follows:

[0006] In a first aspect, an epigenetic gene editing system based on CRISPR / dCas12a is provided, wherein the epigenetic gene editing system comprises: a superCRISPRon fusion protein and a crRNA that guides the superCRISPRon fusion protein to target a target gene; the amino acid sequence of the superCRISPRon fusion protein is shown in any one of SEQ ID NO.1~6.

[0007] A preferred technical solution is that the crRNA is shown in any one of SEQ ID NO.7~13.

[0008] In a preferred technical solution, the target gene is snrpn, RANKL, MMP2, MAGEB2 or FSTN.

[0009] In a second aspect, an application of the epigenetic gene editing system as described in the first aspect is provided, wherein the epigenetic gene editing system is applied to activate the expression of a target gene in a cell.

[0010] A preferred technical solution is to apply the epigenetic gene editing system to activate the expression of FSTN in muscle satellite cells to promote cell differentiation.

[0011] A preferred technical solution is a method for applying the epigenetic gene editing system to activate the expression of a target gene in a cell, comprising the steps of:

[0012] A superCRISPRon vector expressing a superCRISPRon fusion protein and a crRNA vector expressing crRNA are constructed respectively;

[0013] The superCRISPRon vector and the crRNA vector are co-transfected into cells, so that the cells express the superCRISPRon fusion protein and crRNA, thereby activating the expression of the target gene in the cells.

[0014] In a preferred technical solution, the cells are HEK293T cells or muscle satellite cells.

[0015] Beneficial effects: The present invention fuses dCas12a protein with DNA demethylase TET1, transcription activator VP64-p65-Rta, and H3K9me3 demethylase KDM4D to obtain superCRISPRon fusion protein, and then designs crRNA to guide its targeting target gene according to the superCRISPRon fusion protein, thereby obtaining an epigenetic gene editing system based on CRISPR / dCas12a. Compared with TET1-dCas12a and TET1-dCas12a-VPR, the epigenetic gene editing system of the present invention has the characteristics of high efficiency and lasting effect. In addition, the epigenetic gene editing system of the present invention can activate the expression of follistatin (FSTN) in muscle satellite cells, improve the differentiation ability of muscle satellite cells, and thus promote muscle growth. The epigenetic gene editing system upgrades the existing tools, improves the editing efficiency and duration, and plays an important role in promoting the application of gene editing in clinical treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a schematic diagram of the principle of the epigenetic gene editing system of the present invention.

[0017] Figure 2 : is a diagram of the experimental results of Example 1 of the present invention; wherein A is a schematic diagram of superCRISPRon-V1 vector, superCRISPRon-V2 vector and superCRISPRon-V3 vector; B is a bar graph of GFP activation levels after transfection of V1 group, V2 group and V3 group; C is a flow cytometry analysis of GFP fluorescence intensity after transfection of V1 group, V2 group and V3 group; D is a schematic diagram of superCRISPRon-V4 vector, superCRISPRon-V5 vector and superCRISPRon-V6 vector; E is a bar graph of GFP activation levels after transfection of V4 group, V5 group and V6 group; F is a flow cytometry analysis of GFP fluorescence intensity after transfection of V4 group, V5 group and V6 group.

[0018] Figure 3: is a diagram of the experimental results of Example 2 of the present invention; wherein A is a schematic diagram of the TET1-dCas12a vector, the TET1-dCas12a-VPR vector and the superCRISPRon-V6 vector; B is a line graph of the activation level of GFP with increasing generations after transfection in the TET1-dCas12a group, the TET1-dCas12a-VPR group and the superCRISPRon-V6 group; C is a line graph of the activation level of GFP in the TET1-dCas12a group, the TET1-dCas12a-VPR group and the superCRISPRon-V6 group. Flow cytometry analysis of the fluorescence intensity of GFP at different times after transfection in the PR group and superCRISPRon-V6 group; D is a photograph of the fluorescence intensity of GFP at different times after transfection in the TET1-dCas12a group, TET1-dCas12a-VPR group and superCRISPRon-V6 group; E is a graph of the gene activation results targeting different gene sites in the TET1-dCas12a-VPR group and superCRISPRon-V6 group on the 4th and 14th days after transfection.

[0019] Figure 4 : is a graph of the experimental results of Example 3 of the present invention; wherein A is a comparison of the efficiency of FSTN crRNA-1, FSTN crRNA-2, and FSTN crRNA-3; B is a graph of FSTN in the superCRISPRon-V6 group and the TET1-dCas12a-VPR group on the 4th and 14th days after transfection Comparison of mRNA expression levels; C is a comparison of FSTN protein expression levels between the superCRISPRon-V6 group and the TET1-dCas12a-VPR group on the 4th and 14th days after transfection; D is a comparison of DNA methylation at CpG sites in the FSTN promoter region between the superCRISPRon-V6 group and the TET1-dCas12a-VPR group on the 4th and 14th days after transfection; E is a comparison of H3K9me3 levels in the FSTN promoter region between the superCRISPRon-V6 group and the TET1-dCas12a-VPR group; F is a comparison of the results of superCRISPRon-V6 and TET1-dCas12a-VPR groups promoting muscle satellite cell differentiation on the 4th and 7th days after transfection. DETAILED DESCRIPTION

[0020] The present invention provides an epigenetic gene editing system based on CRISPR / dCas12a and its application. In order to make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described below through specific embodiments.

[0021] The CRISPRon vector (catalog number: #167983), U6-crRNA vector (catalog number: #78742), pcDNA-flag-mKdm4d-polyA vector (catalog number: #61553), and dCas12a-VPR vector (catalog number: #188513) used in the present invention were all purchased from Addgene, and the restriction endonucleases KpnⅠ (catalog number: ER0521), AscⅠ (catalog number: ER1891), SphⅠ (catalog number: FD0604), SbfⅠ (catalog number: ER1191), Esp3I (catalog number: ER0451), and MluⅠ (catalog number: ER0561) used were all purchased from Thermo.

[0022] Example 1: Construction of epigenetic gene editing system and efficiency verification

[0023] (1) Design of epigenetic gene editing system

[0024] On the basis of the dCas12a-VPR vector, the nucleotide sequence of the histone H3K9 demethylase KDM4D and the nucleotide sequence of the DNA demethylase TET1 were inserted to obtain the superCRISPRon vector. The superCRISPRon vector expresses the superCRISPRon fusion protein including the histone H3K9 demethylase KDM4D, the DNA demethylase TET1, the dCas12a protein and the transcription activator VPR. The superCRISPRon fusion protein and the crRNA that guides it to target the target gene constitute an epigenetic gene editing system to regulate the target gene. The specific principle is as follows Figure 1 shown.

[0025] (2) Construction of superCRISPRon vector

[0026] According to the different locations of KDM4D, there are three types of superCRISPRon vectors: superCRISPRon-V1 vector, superCRISPRon-V2 vector and superCRISPRon-V3 vector, as follows:

[0027] The DNA fragment encoding KDM4D as shown in SEQ ID NO.14 was obtained from the pcDNA-flag-mKdm4d-polyA vector by polymerase chain reaction (PCR), and the DNA fragment encoding TET1 as shown in SEQ ID NO.15 was obtained from the CRISPRon vector by PCR. Gel recovery was performed, the target band was cut, gel recovery and purification were performed, and the two fragments were connected by overlap PCR to obtain the DNA fragment encoding KDM4D and TET1, which was recorded as KDM4D-TET1. The dCas12a-VPR vector was digested at 37℃ for 1 hour using KpnⅠ and AscⅠ, and then gel recovery was performed and gel recovery and purification were performed. Using the Tiangen homologous recombination kit, KDM4D-TET1 was connected to the linearized dCas12a-VPR vector at 50℃ for 15 minutes, transformed, picked, and sent to Sangon for sequencing to obtain the superCRISPRon-V1 vector. The superCRISPRon-V1 vector expresses the superCRISPRon fusion protein as shown in SEQ ID NO.1.

[0028] The dCas12a-VPR vector was digested at 37°C for 1 hour using KpnⅠ and AscⅠ, and then gel-recovered and gel-purified. Using the Tiangen homologous recombination kit, the DNA fragment encoding TET1 was connected to the linearized dCas12a-VPR vector at 50°C for 15 minutes, transformed, picked, and sent to Sangon for sequencing to obtain the TET1-dCas12a-VPR vector. The TET1-dCas12a-VPR vector was digested at 37°C for 1 hour using SphⅠ and SbfⅠ, and then gel-recovered and gel-purified. Using the Tiangen homologous recombination kit, the DNA fragment encoding KDM4D was connected to the linearized TET1-dCas12a-VPR vector at 50°C for 15 minutes, transformed, picked, and sent to Sangon for sequencing to obtain the superCRISPRon-V2 vector. The superCRISPRon-V2 vector expresses the superCRISPRon fusion protein shown in SEQ ID NO.2.

[0029] The superCRISPRon-V1 vector was digested with KpnⅠ and AscⅠ at 37℃ for 1 hour, and then gel-recovered and purified. The DNA fragment encoding KDM4D was connected to the linearized superCRISPRon-V1 vector at 50℃ for 15 minutes using the Tiangen homologous recombination kit, transformed, picked, and sent to Sangon for sequencing to obtain the superCRISPRon-V3 vector. The superCRISPRon-V3 vector expresses the superCRISPRon fusion protein shown in SEQ ID NO.3.

[0030] (3) Construction of U6-snrpn crRNA vector

[0031] Design a crRNA targeting snrpn, denoted as snrpn crRNA, and its nucleotide sequence is shown in SEQ ID NO.7. Synthesize a DNA fragment encoding snrpn crRNA from TaKaRa. Use Esp3I to digest the U6-crRNA vector at 37°C for 1 hour, then perform gel recovery and gel recovery purification. Use TaKaRa's Solution Ⅰ reagent to connect the DNA fragment encoding snrpncrRNA to the linearized U6-crRNA vector at 16°C overnight, transform, pick bacteria, and send to Sangon for sequencing to obtain the U6-snrpn crRNA vector.

[0032] (4) Efficiency verification of epigenetic gene editing system

[0033] The superCRISPRon-V1 vector, superCRISPRon-V2 vector, and superCRISPRon-V3 vector were transfected with the U6-snrpn crRNA vector into HEK293T cells stably expressing Dazl-snrpn-GFP, respectively, and were recorded as V1, V2, and V3 groups; HEK293T cells without vector transfection were the control group. Dazl in HEK293T cells is highly methylated, so the subsequent GFP green fluorescence is not expressed. The ratio of green fluorescent protein (GFP) positive cells was detected by flow cytometry. The experimental results are shown in Figure 2 As shown in A~C. Figure 2 As shown in A to C, the V1 group transfected with superCRISPRon-V1 vector and U6-snrpn crRNA vector had the highest efficiency of reactivating GFP expression, reaching 74.4%. Therefore, the superCRISPRon-V1 vector was selected for further optimization.

[0034] (5) Optimizing superCRISPRon vectors

[0035] Three linker sequences were synthesized from Sangon: XTEN16, XTEN80, and (GGS)5; the nucleotide sequences of XTEN16 and XTEN80 are shown in reference 1; the nucleotide sequence of (GGS)5 is shown in reference 2; the three linker sequences were connected to the DNA fragment encoding KDM4D by PCR, and then connected to the DNA fragment encoding TET1 to obtain three KDM4D-linker-TET1. The dCas12a-VPR vector was digested at 37℃ for 1 hour using KpnⅠ and AscⅠ, and then gel-recovered and purified by gel-recovery. Using the Tiangen homologous recombination kit, the three KDM4D-linker-TET1 were connected to the linearized dCas12a-VPR vector at 50℃ for 15 minutes, transformed, picked, and sent to Sangon for sequencing to obtain the superCRISPRon-V4 vector containing XTEN16, the superCRISPRon-V5 vector containing XTEN80, and the superCRISPRon-V6 vector containing (GGS)5. The superCRISPRon-V4 vector expresses the superCRISPRon fusion protein as shown in SEQ ID NO.4, the superCRISPRon-V5 vector expresses the superCRISPRon fusion protein as shown in SEQ ID NO.5, and the superCRISPRon-V6 vector expresses the superCRISPRon fusion protein as shown in SEQ ID NO.6.

[0036] Literature 1: Schellenberger V, Wang CW, Geething NC, Spink BJ, Campbell A,To W, Scholle MD, Yin Y, Yao Y, Bogin O, Cleland JL, Silverman J, Stemmer WP.A recombinant polypeptide extends the in vivo half-life of peptides andproteins in a tunable manner. Nat Biotechnol. 2009 Dec;27(12):1186-90. doi:10.1038 / nbt.1588. PMID: 19915550.

[0037] Literature 2: O'Geen H, Ren C, Nicolet CM, Perez AA, Halmai J, Le VM, MackayJP, Farnham PJ, Segal DJ. dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression. NucleicAcids Res. 2017 Sep 29;45(17):9901-9916. doi: 10.1093 / nar / gkx578. PMID:28973434; PMCID: PMC5622328.

[0038] (6) Efficiency verification of the optimized epigenetic gene editing system

[0039] The superCRISPRon-V4 vector, superCRISPRon-V5 vector and superCRISPRon-V6 vector were transfected with the U6-snrpn crRNA vector into HEK293T cells stably expressing Dazl-snrpn-GFP, respectively, and were recorded as V4, V5 and V6 groups; HEK293T cells without vector transfection were the control group; the GFP-positive cell ratio was detected by flow cytometry. The experimental results are shown in Figure 2 As shown in D~F. Figure 2 As shown in D~F, the V6 group transfected with superCRISPRon-V6 vector and U6-snrpn crRNA vector had the highest efficiency of reactivating GFP expression, reaching 75.8%. Therefore, the superCRISPRon-V6 vector was selected for subsequent experiments.

[0040] Example 2: Comparison of the efficiency of the epigenetic gene editing system of Example 1 with existing tools

[0041] (1) Comparison of the efficiency of activating exogenous gene expression

[0042] The TET1-dCas12a-VPR vector was digested at 37°C for 1 hour using MluⅠ and SphⅠ to remove VPR, and then gel-recovered. The TET1-dCas12a vector fragment with VPR removed was gel-recovered and purified. The P2A sequence was synthesized from Sangon as a connection fragment, and its nucleotide sequence is shown in SEQ ID NO.16. Using the Tiangen homologous recombination kit, the P2A sequence was connected to the TET1-dCas12a vector fragment at 50°C for 15 minutes, transformed, picked, and sent to Sangon for sequencing to obtain the TET1-dCas12a vector.

[0043] The TET1-dCas12a vector, TET1-dCas12a-VPR vector and superCRISPRon-V6 vector were transfected with the U6-snrpn crRNA vector prepared in Example 1 into HEK293T cells stably expressing Dazl-snrpn-GFP, and recorded as TET1-dCas12a group, TET1-dCas12a-VPR group and superCRISPRon-V6 group; HEK293T cells not transfected with the vector were used as the control group. Green fluorescence photos of the 0th, 10th, 20th, 30th and 40th generations were taken using a fluorescence microscope, and the GFP-positive cell ratio was detected by flow cytometry. The experimental results are shown in Figure 2. Figure 3 As shown in A~D. Figure 3 As can be seen from A to D, compared with the TET1-dCas12a group and the TET1-dCas12a-VPR group, the superCRISPRon-V6 group has a higher efficiency in improving GFP expression and can be maintained up to 40 generations.

[0044] (2) Comparison of the efficiency of activating endogenous gene expression

[0045] The crRNA targeting RANKL was designed, denoted as RANKL crRNA, and its nucleotide sequence was shown in SEQ ID NO.8; the crRNA targeting MMP2 was denoted as MMP2 crRNA, and its nucleotide sequence was shown in SEQ ID NO.9; the crRNA targeting MAGEB2 was denoted as MAGEB2 crRNA, and its nucleotide sequence was shown in SEQ ID NO.10. The U6-RANKL crRNA vector expressing RANKL crRNA, the U6-MMP2 crRNA vector expressing MMP2 crRNA, and the U6-MAGEB2 crRNA vector expressing MAGEB2 crRNA were constructed, and the specific method was the same as the method for constructing the U6-snrpn crRNA vector in Example 1.

[0046] The U6-RANKL crRNA vector, U6-MMP2 crRNA vector and U6-MAGEB2 crRNA vector were transfected into HEK293T cells with the superCRISPRon-V6 vector constructed in Example 1, respectively, and recorded as the superCRISPRon-V6 group; the U6-RANKL crRNA vector, U6-MMP2 crRNA vector and U6-MAGEB2 crRNA vector were transfected into HEK293T cells with the TET1-dCas12a-VPR vector, respectively, and recorded as the TET1-dCas12a-VPR group; the superCRISPRon-V6 vector or TET1-dCas12a-VPR vector constructed in Example 1 was transfected into HEK293T cells with the U6-crRNA vector, respectively, and recorded as the blank group; HEK293T cells not transfected with the vector were used as the control group. The expression levels of MYL6, DYNCLI1, and LAMP2 were detected by quantitative polymerase chain reaction (qPCR) on the 4th and 14th days, respectively. Figure 3 As shown in E. Figure 3 As shown in Figure E, on the 4th day, the activation efficiency of the superCRISPRon-V6 group was higher than that of the TET1-dCas12a-VPR group, reaching 10 to 80 times; on the 14th day, the activation efficiency of the superCRISPRon-V6 group was still maintained at 5 to 70 times, while the activation efficiency of the TET1-dCas12a-VPR group was significantly reduced.

[0047] Example 3: Promoting muscle satellite cell differentiation using the epigenetic gene editing system of Example 1

[0048] Three crRNAs targeting FSTN were designed, denoted as FSTN crRNA-1, FSTN crRNA-2 and FSTN crRNA-3. The nucleotide sequence of FSTN crRNA-1 is shown in SEQ ID NO.11, the nucleotide sequence of FSTN crRNA-2 is shown in SEQ ID NO.12, and the nucleotide sequence of FSTN crRNA-3 is shown in SEQ ID NO.13. U6-FSTN crRNA-1 vector expressing FSTN crRNA-1, U6-FSTN crRNA-2 vector expressing FSTN crRNA-2 and U6-FSTN crRNA-3 vector expressing FSTN crRNA-3 were constructed, and the specific method was the same as the method for constructing the U6-snrpn crRNA vector in Example 1.

[0049] The U6-FSTN crRNA-1 vector, U6-FSTN crRNA-2 vector, and U6-FSTN crRNA-3 vector were transfected with the superCRISPRon-V6 vector constructed in Example 1 into muscle satellite cells, respectively, and recorded as FSTN-1 group, FSTN-2 group, and FSTN-3 group; cells not transfected with the vector were the control group. The crRNA with the highest efficiency was screened by qPCR, and the experimental results are shown in Figure 4 As shown in A. Figure 4 As shown in Figure A, the FSTN-2 group transfected with U6-FSTN crRNA-2 vector and superCRISPRon-V6 vector had the highest activation efficiency.

[0050] The superCRISPRon-V6 vector and TET1-dCas12a-VPR vector constructed in Example 1 were transfected into muscle satellite cells with the U6-FSTN crRNA-2 vector, respectively, and recorded as the superCRISPRon-V6 group and the TET1-dCas12a-VPR group; cells not transfected with the vector were the control group. qPCR was used to detect changes in FSTN mRNA expression levels on the 4th and 14th days, Western Blot was used to detect changes in FSTN protein expression levels, sulfite sequencing was used to detect changes in DNA methylation in the FSTN promoter region, and chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) was used to detect HEK9me3 deposition in the FSTN promoter region. The experimental results are shown in Figure 2. Figure 4 As shown in B~E. Figure 4 As shown in Figures B to E, compared with the TET1-dCas12a-VPR group, the mRNA expression level and protein expression level of FSTN in the superCRISPRon-V6 group were significantly increased, the DNA methylation in the FSTN promoter region was significantly reduced, and the H3K9me3 deposition in the FSTN promoter region was reduced. After induction of differentiation, MYH2 expression was detected by immunofluorescence on the 4th and 7th days to detect the differentiation of muscle satellite cells. The experimental results are as follows Figure 4 As shown in F. Figure 4 As shown in Figure F, the MYH2 expression level in the superCRISPRon-V6 group was significantly increased, indicating that its differentiation ability was significantly enhanced.

[0051] In summary, the present invention successfully constructed an epigenetic gene editing system based on CRISPR / dCas12a, which includes a superCRISPRon fusion protein and a crRNA that guides the superCRISPRon fusion protein to target the target gene. The system can efficiently and durably activate the expression of the target gene by reducing the DNA methylation level of the target gene, and can significantly improve the differentiation ability of muscle satellite cells when targeting FSTN.

[0052] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. An epigenetic gene editing system based on CRISPR / dCas12a, characterized in that: The epigenetic gene editing system comprises: a superCRISPRon fusion protein and a crRNA that guides the superCRISPRon fusion protein to target a target gene; the amino acid sequence of the superCRISPRon fusion protein is shown in any one of SEQ ID NO.1 or SEQ ID NO.6; The target gene is snrpn, and the crRNA is shown as SEQ ID NO.7; or, the target gene is RANKL, and the crRNA is shown as SEQ ID NO.8; or, the target gene is MMP2, and the crRNA is shown as SEQ ID NO.9; or, the target gene is MAGEB2, and the crRNA is shown as SEQ ID NO.10; or, the target gene is FSTN, and the crRNA is shown as SEQ ID NO.

12.

2. An application of the epigenetic gene editing system as claimed in claim 1, characterized in that: The epigenetic gene editing system is applied to activate the expression of target genes in cells; the target genes are snrpn, RANKL, MMP2, MAGEB2 or FSTN; the application is a non-disease diagnosis and treatment method.

3. The use according to claim 2, characterized in that: The epigenetic gene editing system was applied to activate the expression of FSTN in muscle satellite cells to promote cell differentiation.

4. The use according to claim 2, characterized in that: The method of applying the epigenetic gene editing system to activate the expression of a target gene in a cell comprises the steps of: A superCRISPRon vector expressing a superCRISPRon fusion protein and a crRNA vector expressing crRNA are constructed respectively; The superCRISPRon vector and the crRNA vector are co-transfected into cells, so that the cells express the superCRISPRon fusion protein and crRNA, thereby activating the expression of the target gene in the cells.

5. The use according to claim 4, characterized in that: The cells are HEK293T cells or muscle satellite cells.

Images