An epigenetic gene editing system based on CRISPR / dCas9 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 / dCas9 and its application. The epigenetic gene editing system comprises: a superCRISPRoff fusion protein and an sgRNA that guides the superCRISPRoff fusion protein to target a target gene; the amino acid sequence of the superCRISPRoff fusion protein is shown in any one of SEQ ID NO.1~6. Compared with the existing tools dCas9‑DNMT3A‑3L or CRISPRoff, 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 significantly inhibit the expression of myostatin in muscle satellite cells, improve the differentiation ability of muscle satellite cells, and thus promote muscle growth.

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 / dCas9 and applications thereof. Background Art

[0002] CRISPR / Cas9 is a powerful genome editing system. With the deepening of research, scientists have established a CRISPR / dCas9 system that regulates genome transcription. The establishment of the CRISPR / dCas9 system is based on the discovery of the dCas9 protein. The dCas9 protein has lost its nuclease activity and cannot cut DNA, but it still has DNA binding activity. It can target the target gene under the guidance of sgRNA and carry specific epigenetic regulatory proteins upstream of the target gene to achieve epigenetic regulation of the target gene. The CRISPR / dCas9 system will not change genomic DNA and is highly efficient and safe.

[0003] In the prior art, the dCas9 protein is fused with the DNA methyltransferases DNMT3A and DNMT3L to obtain the epigenetic editor dCas9-DNMT3A-3L. dCas9-DNMT3A-3L can increase the methylation level of the CpG region of the target gene, thereby inhibiting the expression of the target gene. On the contrary, fusing the dCas9 protein with the DNA demethylase TET1 can selectively inhibit the DNA methylation level in the target region, thereby promoting the expression of the target gene. In 2021, Jonathan S. Weissman et al. fused DNMT3A, DNMT3L and the transcriptional repressor KRAB to the N- / C-terminus of dCas9, respectively, to construct a new epigenetic editor CRISPRoff, which can persistently inhibit the expression of the target gene by increasing DNA methylation. However, the transcriptional inhibition efficiency and inhibition maintenance time of the two epigenetic editors, dCas9-DNMT3A-3L or CRISPRoff, still need to be improved.

[0004] Therefore, the prior art still needs to be improved and developed. Summary of the invention

[0005] 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 / dCas9 and its application, aiming to solve the problems of low transcriptional inhibition efficiency and short inhibition maintenance time of the existing epigenetic gene editing system.

[0006] The technical solution of the present invention is as follows:

[0007] In a first aspect, a CRISPR / dCas9-based epigenetic gene editing system is provided, comprising: a superCRISPRoff fusion protein and an sgRNA that guides the superCRISPRoff fusion protein to target a target gene; the amino acid sequence of the superCRISPRoff fusion protein is shown in any one of SEQ ID NO.1~6.

[0008] In a preferred technical solution, the sgRNA is shown in any one of SEQ ID NOs. 7 to 13.

[0009] In a preferred technical solution, the target gene is snrpn, MYL6, DYNCLI1, LAMP2 or MSTN.

[0010] 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 inhibit the expression of a target gene in a cell.

[0011] A preferred technical solution is to apply the epigenetic gene editing system to inhibit the expression of MSTN in muscle satellite cells to promote cell differentiation.

[0012] A preferred technical solution is to apply the epigenetic gene editing system to a method for inhibiting the expression of a target gene in a cell, comprising the steps of:

[0013] A superCRISPRoff vector expressing superCRISPRoff fusion protein and a sgRNA vector expressing sgRNA were constructed respectively;

[0014] The superCRISPRoff vector and the sgRNA vector are co-transfected into cells, so that the cells express the superCRISPRoff fusion protein and sgRNA, thereby inhibiting the expression of the target gene in the cells.

[0015] In a preferred technical solution, the cells are HEK293T cells or muscle satellite cells.

[0016] Beneficial effects: The present invention fuses dCas9 protein with DNA methyltransferase DNMT3A-3L, transcriptional repressor KRAB, and H3K9me3-specific methyltransferase G9A to obtain a superCRISPRoff fusion protein, and then designs sgRNA that guides its targeting of the target gene according to the superCRISPRoff fusion protein to obtain an epigenetic gene editing system based on CRISPR / dCas9. Compared with the existing tools dCas9-DNMT3A-3L or CRISPRoff, 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 significantly inhibit the expression of myostatin (MSTN) 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

[0017] Figure 1 It is a schematic diagram of the principle of the epigenetic gene editing system of the present invention.

[0018] Figure 2 : It is a diagram of the experimental results of Example 1 of the present invention; wherein A is a schematic diagram of the superCRISPRoff-V1 vector, the superCRISPRoff-V2 vector and the superCRISPRoff-V3 vector; B is a bar graph of the GFP inhibition level after transfection of the V1 group, the V2 group and the V3 group; C is a flow cytometry analysis of the GFP fluorescence intensity after transfection of the V1 group, the V2 group and the V3 group; D is a schematic diagram of the superCRISPRoff-V4 vector, the superCRISPRoff-V5 vector and the superCRISPRoff-V6 vector; E is a bar graph of the GFP inhibition level after transfection of the V4 group, the V5 group and the V6 group; F is a flow cytometry analysis of the GFP fluorescence intensity after transfection of the V4 group, the V5 group and the V6 group.

[0019] Figure 3: It is a diagram of the experimental results of Example 2 of the present invention; wherein A is a schematic diagram of the dCas9-DNMT3A-3L vector, the CRISPRoff vector and the superCRISPRoff-V6 vector; B is a line graph of the inhibition level of GFP in the DNMT3A-3L-dCas9 group, the CRISPRoff group and the superCRISPRoff-V6 group after transfection with increasing generations; C is a flow cytometry analysis of the fluorescence intensity of GFP at different times after transfection in the DNMT3A-3L-dCas9 group, the CRISPRoff group and the superCRISPRoff-V6 group; D is a photograph of the fluorescence intensity of GFP at different times after transfection in the DNMT3A-3L-dCas9 group, the CRISPRoff group and the superCRISPRoff-V6 group; E is a diagram of the gene inhibition results of the CRISPRoff group and the superCRISPRoff-V6 group targeting different gene sites on the 4th and 14th days after transfection.

[0020] Figure 4 : is a graph of experimental results related to Example 3 of the present invention; wherein A is a graph comparing the efficiencies of MSTN sgRNA-1, MSTN sgRNA-2, and MSTN sgRNA-3; B is a graph comparing the MSTN mRNA inhibition levels of the superCRISPRoff-V6 group and the CRISPRoff group on the 4th and 14th days after transfection; C is a graph comparing the MSTN protein inhibition levels of the superCRISPRoff-V6 group and the CRISPRoff group on the 4th and 14th days after transfection; D is a graph comparing the DNA methylation of CpG sites in the MSTN promoter region of the superCRISPRoff-V6 group and the CRISPRoff group on the 4th and 14th days after transfection; E is a graph comparing the H3K9me3 levels in the MSTN promoter region of the superCRISPRoff-V6 group and the CRISPRoff group; and F is a graph showing the results of promoting muscle satellite cell differentiation of the superCRISPRoff-V6 group and the CRISPRoff group on the 4th and 7th days after transfection. DETAILED DESCRIPTION

[0021] The present invention provides an epigenetic gene editing system based on CRISPR / dCas9 and applications thereof. In order to make the objectives, technical solutions and effects of the present invention clearer and more specific, the present invention is further described below through specific embodiments.

[0022] The G9A-dCas9 vector (catalog number: #100089), CRISPRoff vector (catalog number: #167981), U6-sgRNA vector (catalog number: #61424), and dCas9-DNMT3A-3L vector (catalog number: #128424) used in the present invention were all purchased from Addgene, and the restriction endonucleases EcoRⅠ (catalog number: ER0271), XmaⅠ (catalog number: ER0172), NotⅠ (catalog number: ER0592), BglⅡ (catalog number: ER0081), and BbsⅠ (catalog number: ER1011) used were all purchased from Thermo.

[0023] Example 1: Construction of epigenetic gene editing system and efficiency verification

[0024] (1) Design of epigenetic gene editing system

[0025] On the basis of CRISPRoff vector, the nucleotide sequence of histone H3K9-specific methyltransferase G9A was inserted to obtain the superCRISPRoff vector. The superCRISPRoff vector expresses the superCRISPRoff fusion protein including histone H3K9-specific methyltransferase G9A, DNA methyltransferase DNMT3A-3L, dCas9 protein and transcription inhibitor KRAB. The superCRISPRoff fusion protein and the sgRNA 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.

[0026] (2) Construction of superCRISPRoff vector

[0027] According to the different positions of G9A, there are three types of superCRISPRoff vectors: superCRISPRoff-V1 vector, superCRISPRoff-V2 vector and superCRISPRoff-V3 vector, as follows:

[0028] The DNA fragment encoding G9A as shown in SEQ ID NO.14 was obtained from the G9A-dCas9 vector by polymerase chain reaction (PCR). Gel recovery was performed, the target band was cut, and gel recovery and purification were performed. The CRISPRoff vector was digested at 37℃ for 1 hour using EcoRⅠ and XmaⅠ, and then gel recovery and gel recovery were performed. Using the Tiangen homologous recombination kit, the DNA fragment encoding G9A was connected to the linearized CRISPRoff vector at 50℃ for 15 minutes, transformed, picked, and sent to Sangon for sequencing to obtain the superCRISPRoff-V1 vector. The superCRISPRoff-V1 vector expresses the superCRISPRoff fusion protein as shown in SEQ ID NO.1.

[0029] The CRISPRoff vector was digested with NotⅠ and BglⅡ at 37℃ for 1 hour, and then gel-recovered and purified. The DNA fragment encoding G9A was connected to the linearized CRISPRoff vector at 50℃ for 15 minutes using the Tiangen homologous recombination kit, transformed, picked, and sent to Sangon for sequencing to obtain the superCRISPRoff-V2 vector. The superCRISPRoff-V2 vector expresses the superCRISPRoff fusion protein shown in SEQ ID NO.2.

[0030] The superCRISPRoff-V1 vector was digested with NotⅠ and BglⅡ at 37℃ for 1 hour, and then gel-recovered and purified. The DNA fragment encoding G9A was connected to the linearized superCRISPRoff-V1 vector at 50℃ for 15 minutes using the Tiangen homologous recombination kit, transformed, picked, and sent to Sangon for sequencing to obtain the superCRISPRoff-V3 vector. The superCRISPRoff-V3 vector expresses the superCRISPRoff fusion protein shown in SEQ ID NO.3.

[0031] (3) Construction of U6-snrpn sgRNA vector

[0032] The sgRNA targeting snrpn was designed and recorded as snrpn sgRNA, and its nucleotide sequence is shown in SEQ ID NO.7. The DNA fragment encoding snrpn sgRNA was synthesized from TaKaRa. The U6-sgRNA vector was digested with BbsⅠ at 37℃ for 1 hour, followed by gel recovery and gel recovery purification. The DNA fragment encoding snrpnsgRNA was connected to the linearized U6-sgRNA vector at 16℃ overnight using TaKaRa's Solution Ⅰ reagent, transformed, picked, and sent to Sangon for sequencing to obtain the U6-snrpn sgRNA vector.

[0033] (4) Efficiency verification of epigenetic gene editing system

[0034] The superCRISPRoff-V1 vector, superCRISPRoff-V2 vector and superCRISPRoff-V3 vector were transfected with the U6-snrpn sgRNA vector into HEK293T cells stably expressing glyceraldehyde-3-phosphate dehydrogenase-small nucleolar RNA processing protein-green fluorescent protein fusion protein (GAPDH-snrpn-GFP), respectively, and were recorded as V1, V2 and V3 groups; HEK293T cells without vector transfection were the control group; the ratio of green fluorescent protein (GFP) negative cells was detected by flow cytometry. The experimental results are shown in Figure 2 As shown in A~C. Figure 2 As shown in Figures A to C, the V1 group transfected with the superCRISPRoff-V1 vector and U6-snrpn sgRNA vector had the highest inhibition efficiency, reaching 76.9%. Therefore, the superCRISPRoff-V1 vector was selected for further optimization.

[0035] (5) Optimizing superCRISPRoff vector

[0036] Three linker sequences were synthesized from Sangon: XTEN16, XTEN80, and (GGS)5; the nucleotide sequences of XTEN16 and XTEN80 are shown in reference 1, and the nucleotide sequence of (GGS)5 is shown in reference 2. The three linker sequences were connected to the DNA fragment encoding G9A by PCR to obtain three G9A-linkers. The CRISPRoff vector was digested at 37°C for 1 hour using EcoRⅠ and XmaⅠ, and then gel-recovered and gel-recovered and purified. Using the Tiangen homologous recombination kit, the three G9A-linkers were connected to the linearized CRISPRoff vector at 50°C for 15 minutes, transformed, picked, and sent to Sangon for sequencing to obtain the superCRISPRoff-V4 vector containing XTEN16, the superCRISPRoff-V5 vector containing XTEN80, and the superCRISPRoff-V6 vector containing (GGS)5. The superCRISPRoff-V4 vector expresses the superCRISPRoff fusion protein as shown in SEQ ID NO.4, the superCRISPRoff-V5 vector expresses the superCRISPRoff fusion protein as shown in SEQ ID NO.5, and the superCRISPRoff-V6 vector expresses the superCRISPRoff fusion protein as shown in SEQ ID NO.6.

[0037] 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.

[0038] 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.

[0039] (6) Efficiency verification of the optimized epigenetic gene editing system

[0040] The superCRISPRoff-V4 vector, superCRISPRoff-V5 vector and superCRISPRoff-V6 vector were transfected with the U6-snrpn sgRNA vector into HEK293T cells stably expressing GAPDH-snrpn-GFP, respectively, and were recorded as V4, V5 and V6 groups; HEK293T cells without vector transfection were the control group; the ratio of GFP-negative cells was detected by flow cytometry. The experimental results are shown in Figure 2 As shown in D~F. Figure 2 As shown in Figures D to F, the V6 group transfected with the superCRISPRoff-V6 vector and U6-snrpn sgRNA vector had the highest inhibition efficiency, reaching 77.4%. Therefore, the superCRISPRoff-V6 vector was selected for subsequent experiments.

[0041] Example 2: Comparison of the efficiency of the epigenetic gene editing system of Example 1 with existing tools

[0042] (1) Comparison of the efficiency of inhibiting exogenous gene expression

[0043] The dCas9-DNMT3A-3L vector, CRISPRoff vector and superCRISPRoff-V6 vector constructed in Example 1 were transfected with the U6-snrpn sgRNA vector prepared in Example 1 into HEK293T cells stably expressing GAPDH-snrpn-GFP, and recorded as DNMT3A-3L-dCas9 group, CRISPRoff group and superCRISPRoff-V6 group; HEK293T cells not transfected with the vector were the control group. Green fluorescence photos of the 0th, 10th, 20th, 30th and 40th generations were taken using a fluorescence microscope, and the ratio of GFP-negative cells was detected using a flow cytometer. The experimental results are shown in Figure 2. Figure 3 As shown in A~D. Figure 3 As shown in A~D, compared with the DNMT3A-3L-dCas9 group and the CRISPRoff group, the superCRISPRoff-V6 group was more efficient in inhibiting GFP expression and could be maintained up to 40 generations.

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

[0045] The sgRNA targeting MYL6 was designed, denoted as MYL6 sgRNA, and its nucleotide sequence was shown in SEQ ID NO.8; the sgRNA targeting DYNCLI1 was denoted as DYNCLI1 sgRNA, and its nucleotide sequence was shown in SEQ ID NO.9; the sgRNA targeting LAMP2 was denoted as LAMP2 sgRNA, and its nucleotide sequence was shown in SEQ ID NO.10. U6-MYL6 sgRNA vector expressing MYL6 sgRNA, U6-DYNCLI1 sgRNA vector expressing DYNCLI1 sgRNA, and U6-LAMP2 sgRNA vector expressing U6-LAMP2 sgRNA were constructed, and the specific method was the same as the method for constructing the U6-snrpn sgRNA vector in Example 1.

[0046] The U6-MYL6 sgRNA vector, U6-DYNCLI1 sgRNA vector and U6-LAMP2 sgRNA vector were transfected into HEK293T cells with the superCRISPRoff-V6 vector constructed in Example 1, respectively, and recorded as the superCRISPRoff-V6 group; the U6-MYL6 sgRNA vector, U6-DYNCLI1 sgRNA vector and U6-LAMP2 sgRNA vector were transfected into HEK293T cells with the CRISPRoff vector, respectively, and recorded as the CRISPRoff group; the superCRISPRoff-V6 vector or CRISPRoff vector constructed in Example 1 was transfected into HEK293T cells with the U6-sgRNA vector, respectively, as the blank group; HEK293T cells not transfected with the vector were the control group. The expression levels of MYL6, DYNCLI1 and LAMP2 were detected by quantitative polymerase chain reaction (qPCR) on the 4th day and the 14th day, respectively. The experimental results are shown in Figure 2. Figure 3 As shown in E. Figure 3 As shown in Figure E, on the 4th day, the inhibition efficiency of the superCRISPRoff-V6 group was higher than that of the CRISPRoff group, reaching 60%~80%; on the 14th day, the inhibition efficiency of the superCRISPRoff-V6 group was still maintained at 40%~70%, while the inhibition efficiency of the CRISPRoff group was significantly reduced.

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

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

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

[0050] The superCRISPRoff-V6 vector and CRISPRoff vector constructed in Example 1 were transfected with the U6-MSTNsgRNA-3 vector into muscle satellite cells, which were recorded as the superCRISPRoff-V6 group and the CRISPRoff group; cells not transfected with the vector were the control group. qPCR was used to detect the changes in MSTN mRNA expression levels on the 4th day and the 14th day, Western Blot was used to detect the changes in MSTN protein expression levels, sulfite sequencing was used to detect the changes in DNA methylation in the MSTN promoter region, and chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) was used to detect the deposition of HEK9me3 in the MSTN 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 CRISPRoff group and the control group, the mRNA expression level and protein expression level of MSTN in the superCRISPRoff-V6 group were significantly decreased, the DNA methylation in the MSTN promoter region was significantly increased, and the H3K9me3 deposition in the MSTN promoter region was increased. After induction of differentiation, the expression of MYH2 was detected by immunofluorescence on the 4th and 7th days, respectively, to detect the differentiation of muscle satellite cells into myotubes. The experimental results are as follows Figure 4 As shown in F. Figure 4 As shown in Figure F, after differentiation induction, the expression level of MYH2 in the superCRISPRoff-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 / dCas9, which includes a superCRISPRoff fusion protein and an sgRNA that guides the superCRISPRoff fusion protein to target the target gene. The system can efficiently and durably inhibit the expression of the target gene by increasing the DNA methylation level of the target gene, and can significantly improve the differentiation ability of muscle satellite cells when targeting MSTN.

[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 / dCas9, characterized in that: The epigenetic gene editing system comprises: a superCRISPRoff fusion protein and an sgRNA that guides the superCRISPRoff fusion protein to target a target gene; the amino acid sequence of the superCRISPRoff fusion protein is shown in SEQ ID NO.1 or SEQ ID NO.

6.

2. The epigenetic gene editing system according to claim 1, characterized in that: The sgRNA is shown in any one of SEQ ID NO.7~13.

3. The epigenetic gene editing system according to claim 1, characterized in that: The target gene is snrpn, MYL6, DYNCLI1, LAMP2 or MSTN.

4. An application of the epigenetic gene editing system according to any one of claims 1 to 3, characterized in that: The epigenetic gene editing system is applied to inhibit the expression of target genes in cells, and the application is a non-disease diagnosis and treatment method.

5. The use according to claim 4, characterized in that: The epigenetic gene editing system was applied to inhibit the expression of MSTN in muscle satellite cells to promote cell differentiation.

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

7. The use according to claim 6, characterized in that: The cells are HEK293T cells or muscle satellite cells.

Images