DNA directional demethylation double lentivirus plasmid system as well as construction method and application thereof

Abstract

The invention relates to the technical field of gene engineering, in particular to a DNA (Deoxyribonucleic Acid) oriented demethylation double lentivirus plasmid system as well as a construction method and application thereof. The double lentivirus plasmid system comprises a dCas9-GCN4s sequence and an Anti-GCN4-scFv-TET1 sequence, and the dCas9-GCN4s sequence and the Anti-GCN4-scFv-TET1 sequence are The sequence of the dCas9-GCN4s is as shown in SEQ ID (sequence identifier number) No.36; the sequence of the Anti-GCN4-scFv-GFP-TET1CD is as shown in SEQ ID No. 37, and the sequence of the Anti-GCN4-scFv-GFP-TET1CD is as shown in SEQ A guidance system and a demethylation system are respectively inserted into a lentiviral vector by means of molecular biology, and are associated by an antigen-antibody technology, and the demethylation effect is amplified, so that an obvious demethylation effect is finally achieved on a target position of a target cell genome.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to a double lentiviral plasmid system for DNA directed demethylation, and a construction method and application thereof. Background Art

[0002] Lentivirus is a type of retrovirus that can integrate its own genome into the host genome after infecting host cells. Lentivirus vectors are based on the lentivirus genome. Through molecular biological methods, virulence-related genes are deleted, and its elements are modified to introduce exogenous target genes and expression elements. They are prepared to be able to carry exogenous genes into host cells and integrate exogenous genes into the host cell genome to achieve their stable expression in host cells. Among them, gene therapy vectors developed based on human immunodeficiency virus-1 (HIV-1) are the most widely used and have many advantages in gene transfection, such as infection efficiency that does not depend on cell division ability, low immunogenicity, and stable and continuous expression.

[0003] The CRISPR-Cas system, namely the "clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins" system, is an efficient gene editing system, which mainly includes single-stranded guide RNA (sgRNA) and Cas endonucleases (including Cas9 and Cas12, etc.). In this system, sgRNA guides Cas endonucleases to specific sites in the genome through complementary base pairing, thereby exerting endonuclease activity to cut the double-stranded DNA at the target site; on this basis, the specific structural domain of the Cas9 protein is mutated to lose its DNA cutting activity, and the resulting dCas9 combined with sgRNA can be regarded as a precise guidance system, which is then fused and expressed with specific enzymes through gene fusion expression technology, such as DNA demethylases such as TET1, etc., to achieve epigenetic editing of specific DNA regions under the guidance of sgRNA.

[0004] With the development of epigenetics, efficient epigenetic editing tools are becoming increasingly important. However, due to the limitation of gene length, ordinary dCas9-TET1 fusion plasmids have low transfection efficiency and poor editing effect. How to improve the methylation removal ability of this system is a key issue that needs to be solved. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a double lentiviral plasmid system for DNA directed demethylation and a construction method and application thereof.

[0006] A dual lentiviral plasmid system for DNA directed demethylation, the dual lentiviral plasmid system comprising a dCas9-GCN4s sequence and an Anti-GCN4-scFv-TET1 sequence; The dCas9-GCN4s sequence is shown in SEQ ID No.36; The Anti-GCN4-scFv-GFP-TET1CD sequence is shown in SEQ ID No.37.

[0007] Preferably, the dual lentiviral plasmid system further comprises an sgRNA targeting a highly methylated region.

[0008] Preferably, the nucleotide sequence of the sgRNA targeting the hypermethylated region is shown in SEQ ID NO.32.

[0009] The method for constructing the dual lentiviral plasmid system comprises the following steps: Insert the dCas9-GCN4s sequence into the downstream of the CMV promoter of the pLVX-IRES-Puro lentiviral plasmid vector to obtain the lentiviral plasmid pLVX-IRES-Puro-dCas9-GCN4s; Insert the Anti-GCN4-scFv-GFP-TET1CD sequence into the downstream of the CMV promoter of the pLVX-IRES-Neo lentiviral plasmid vector to obtain the lentiviral plasmid pLVX-IRES-Neo-Anti-GCN4-scFv-GFP-TET1CD; The lentiviral plasmid pLVX-IRES-Puro-dCas9-GCN4s is packaged to obtain lentiviral solution 1, and the lentiviral plasmid pLVX-IRES-Neo-Anti-GCN4-scFv-GFP-TET1CD is packaged to obtain lentiviral solution 2. The lentiviral solution 1 and the lentiviral solution 2 are used to infect the same cell, and sgRNA targeting the highly methylated region is introduced to obtain the dual lentiviral plasmid system.

[0010] Preferably, the cell is human lung adenocarcinoma cell PC-9.

[0011] Preferably, the cell is human glioma cell U251.

[0012] Preferably, human embryonic kidney 293T cells are used for packaging.

[0013] Preferably, the nucleotide sequence of the sgRNA targeting the hypermethylated region is shown in SEQ ID NO.32.

[0014] The double lentiviral plasmid system is used in the production of lentiviral plasmids for DNA directed demethylation.

[0015] The dual lentiviral plasmid system is used in the directed demethylation of eukaryotic cell DNA.

[0016] Application of lentivirus in achieving targeted demethylation of DNA in eukaryotic cells.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a dual lentiviral system, which can directionally remove methylation in a specified region of eukaryotic cell DNA, thereby improving the methylation removal effect.

[0018] The present invention inserts the guide system and the demethylation system into the lentiviral vector respectively by molecular biological means, and associates the two and amplifies the demethylation effect by antigen-antibody technology, and finally plays a significant demethylation effect on the target position of the target cell genome.

[0019] This system can provide a reference for the research tools of various epigenetic modifications such as acetylation and glycosylation of DNA and histones, which is of great significance for epigenetic research. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The following is a schematic diagram of the experimental process for the construction and use of the tool, where ① is the lentiviral plasmid pLVX-IRES-Puro-dCas9-GCN4s, ② is the lentiviral plasmid pLVX-IRES-Neo-Anti-GCN4-scFv-GFP-TET1CD, ③ is the lentivirus-dCas9-GCN4s, and ④ is the lentivirus-pLVX-IRES-Neo-Anti-GCN4-scFv-GFP-TET1CD.

[0021] Figure 2 Schematic diagram of the principle of targeted amplification demethylation, where ① is the lentiviral gene dCas9-GCN4s, and ② is the lentiviral gene Anti-GCN4-scFv-GFP-TET1CD. Figure 3 It is an electrophoresis diagram, among which ① is the result of dCas9-GCN4s fragment, ② is the parallel experimental result of 4 single colonies in which dCas9-GCN4s fragment was successfully connected and recombined with PLVX-IRES-PURO plasmid, ③ is the result of Anti-GCN4-scFv-GFP-TET1CD fragment, ④ is the parallel experimental result of 4 single colonies in which Anti-GCN4-scFv-GFP-TET1CD fragment was successfully connected and recombined with PLVX-IRES-NEO plasmid, and ⑤ is Marker.

[0022] Figure 4 Comparison of PC-9 demethylation effects. Note: Each circle represents the methylation ratio of the methylation site. The black part is the methylation ratio, and the white part is the non-methylation ratio.

[0023] Figure 5 Comparison of the demethylation effect of U251. Note: Each circle represents the methylation ratio of the methylation site, the black part is the methylation ratio, and the white part is the non-methylation ratio.

[0024] Figure 6 The nucleotide sequence of dCas9-GCN4s is shown.

[0025] Figure 7 The nucleotide sequence of Anti-GCN4-scFv-TET1 is shown.

[0026] Figure 8 It is the 1st bp to 4645bp of the sequence of pLVX-IRES-Puro-dCas9-GCN4s.

[0027] Fig. 9 It is 4645bp~9429bp of the sequence of pLVX-IRES-Puro-dCas9-GCN4s.

[0028] Fig.10 It is 9430bp~13015bp of the sequence of pLVX-IRES-Puro-dCas9-GCN4s.

[0029] Figure 8~Figure 10 Sequences that make up pLVX-IRES-Puro-dCas9-GCN4s.

[0030] Fig.11 It is the 1st bp to 5292 bp of the sequence of pLVX-IRES-Neo-Anti-GCN4-scFv-GFP-TET1CD.

[0031] Fig.12 It is 5293bp~11168bp of the sequence of pLVX-IRES-Neo-Anti-GCN4-scFv-GFP-TET1CD.

[0032] Fig.13 It is 11169bp~12267bp of the sequence of pLVX-IRES-Neo-Anti-GCN4-scFv-GFP-TET1CD.

[0033] Figure 11~Figure 13 Composition: pLVX-IRES-Neo-Anti-GCN4-scFv-GFP-TET1CD. DETAILED DESCRIPTION

[0034] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified.

[0035] Experimental Materials: LB liquid medium: peptone 10g, yeast extract 5g, NaCl 10g, add deionized water to 1L, adjust the pH value to 7.0, sterilize at high temperature and high pressure, and store at 4℃; LB solid medium: peptone 10g, yeast extract 5g, NaCl 10g, agar powder 15g, add deionized water to 1L, adjust the pH value to 7.0, sterilize at high temperature and high pressure, cool to 60℃, add antibiotics according to experimental requirements, shake well and pour into a 10cm bacterial culture dish, open the lid and let it air for 2h, seal and store at 4℃ after the plate solidifies; trypsin (catalog number: 15400054, Thermo Fisher); fetal bovine serum (catalog number A5256701, GIBCO); DMEM (catalog number G4511, Sevier); PBS: KH2PO4 0.27g, KCl 0.2g, Na2HPO4·12H2O 3.58g, NaCl 8.0g, add deionized water to 1L, adjust the pH to 7.4; ECORI-XBAI (Cat. No. R0145, Biolab); Kanamycin (Cat. No.: 60206ES, Yisheng Biotechnology Co., Ltd.); Ampicillin (Cat. No.: 60203ES, Yisheng Biotechnology Co., Ltd.); Puromycin (Cat. No.: 60210ES, Yisheng Biotechnology Co., Ltd.); G418 Geneticin (Cat. No.: 60220ES, Yisheng Biotechnology Co., Ltd.); Gel Recovery Kit (Cat. No. NA111-1KT, Merck Sigma); High-fidelity PCR Polymerase (PV2) (Cat. No.: 1401, Peplib); Recombinase (Cat. No.: DLV201, Daling Biotechnology); Plasmid Mini-Pickup Kit (Cat. No. DP103, TIANGEN); Plasmid Large-Pickup Kit (Cat. No. DP117, TIANGEN); Lipofectamine 2000 (Cat. No.: 11668, Thermo Fisher); polybrene (Cat. No. 40804ES, Yisheng Biotechnology Co., Ltd.); genome extraction kit (Cat. No. DP304, TIANGEN); bisulfite conversion kit (Cat. No. 59104, QIAGEN); chemical synthesis of sgRNA (safe edit sgRNA synthesis service of Nanjing GenScript Biotechnology Co., Ltd.); pyrosequencing (Q48 pyrosequencing service of Shanghai Jelan Biotechnology Co., Ltd.). Human embryonic kidney 293T cells; human lung adenocarcinoma cells PC-9; human glioma cells U251 were purchased from Shanghai Cell Bank and preserved in this laboratory.

[0036] The overall experimental process and demethylation principle can be found in Figure 1 , Figure 2 . Figure 2Double lentiviral DNA fragments (red and green double helices) were successfully inserted into the genomic DNA (blue double helix) of the target cells, and dCas9-GCN4 fusion protein and scFv-GFP-TET1CD fusion protein were successfully expressed in the target cells. When the sgRNA targeting the target region is introduced, the sgRNA first assembles with dCas9 and guides it to the target position. The GCN4 on dCas9 then interacts with scFv-GCN4 to recruit multiple TET1CDs to the target region to achieve an amplified demethylation effect on the target region.

[0037] The primers and sgRNA01 targeting sequences used in the present invention are shown in Table 1, the PCR reaction system is shown in Table 2, and the PCR reaction conditions are shown in Table 3.

[0038] The template plasmid 82559 used in the experiment was purchased from addgene, source website: https: / / www.addgene.org / , the sequence of the lentiviral plasmid pLVX-IRES-Puro-dCas9-GCN4s is shown in SEQ ID No.38, and the sequence of the lentiviral plasmid pLVX-IRES-Neo-Anti-GCN4-scFv-GFP-TET1CD is shown in SEQ ID No.39.

[0039] Table 1 Primers and sgRNA01 targeting sequences used in the present invention Table 2 PCR reaction system Table 3 PCR reaction conditions Example 1. Obtaining the template plasmid Use the plasmid No. 82559 on the Addgene website as the original template, transform E. coli DH5α, and obtain sufficient template through shaking, screening, and plasmid extraction. The details are as follows:

[0040] (1) After the competent bacteria DH5α is thawed on ice, take 50 μL and add it to a 1.5 mL centrifuge tube and place it on ice. Take 82559 plasmid with a concentration of 800 ng / μL, take 1 μL and add it to the above DH5α bacterial solution and mix well. Incubate on ice for 30 min and then place in a 42°C water bath for 90 s (do not move). After the end, immediately transfer to an ice-water mixture and let it stand for 2 min. (2) Add 200 μL of LB liquid culture medium to the centrifuge tube and place it on a 37°C constant temperature shaker at 150 rpm for 40 min. (3) Take LB plate culture medium, evenly spread the bacterial solution in the centrifuge tube on the surface of the plate in a clean bench, and then place the culture medium in an incubator at 37°C for 16 hours; (4) After the culture is completed, pick a single clone in the clean bench, culture each colony with 5 mL of LB liquid culture medium containing 20 mg / mL kanamycin, place in a shaker at 37°C, and culture at 220 r / min for 10 h; (5) After the culture is completed, take 2 mL of the bacterial solution from each tube and use the Tiangen Plasmid Extraction Kit (Cat. No. DP103) to extract the plasmid according to the instructions and perform enzyme digestion identification. For the successfully identified strains, add 8 mL of LB liquid culture medium containing 20 mg / mL kanamycin to each tube and place it in a 37°C shaker at 220 r / min for 10 h; then add the resulting bacterial solution to 150 mL of LB liquid culture medium containing 20 mg / mL kanamycin and culture it at 220 r / min for 10 h. Use the Tiangen Plasmid Extraction Kit (Cat. No. DP117) to extract the plasmid from the resulting bacterial solution.

[0041] 2. Connect the target fragment to the lentiviral vector The dCas9-GCN4s fragment and the Anti-GCN4-scFv-GFP-TET1CD fragment were cut from the 82559 plasmid template by PCR amplification, and the target fragment was obtained by electrophoresis and gel recovery. Then, the target fragment and the vector plasmid were digested and connected by restriction endonuclease double digestion method, and then transformed into Escherichia coli, single clone colonies were selected to screen for successful strains, and the corresponding target plasmid was extracted. The dCas9-GCN4s sequence is shown in SEQ ID No.36 and Figure 6 , the Anti-GCN4-scFv-GFP-TET1CD sequence is shown in SEQ ID No.37 and Figure 7 The details are as follows:

[0042] 1. Construction of pLVX-IRES-Puro-dCas9-GCN4s plasmid (1) Using 82559 plasmid as template, a product XA0139130-1A was obtained by PCR reaction. The primer sequences used are shown in SEQ ID NO. 1 and SEQ ID NO. 4 in Table 1, the PCR system is shown in sequence number 1 in Table 2, and the PCR reaction conditions are shown in Table 3; (2) Using 82559 plasmid as template, a second product XA0139130-1B was obtained by PCR reaction. The primer sequences used are shown in SEQ ID NO. 2 and SEQ ID NO. 3 in Table 1, the PCR system is shown in sequence number 2 in Table 2, and the PCR reaction conditions are shown in Table 3; (3) Using XA0139130-1A and XA0139130-1B as templates, a target fragment, dCas9-GCN4s fragment, was obtained by PCR reaction. The primer sequences used are shown in SEQ ID NO. 3 and SEQ ID NO. 4 in Table 1, the PCR system is shown in sequence number 3 in Table 2, and the PCR reaction conditions are shown in Table 3; (4) Gel recovery of target fragment - dCas9-GCN4s fragment (see Figure 3 ①), and treat PLVX-IRES-PURO plasmid with ECORI-XBAI, and perform gel recovery to obtain linearized PLVX-IRES-PURO-ECORI-XBAI vector; 4 μL target fragment 1, 3.5 μL linearized PLVX-IRES-PURO-ECORI-XBAI vector, and 2.5 μL recombinase were mixed, placed in a 50°C water bath for 25 min, placed for 3 min to cool down, and then transformed and coated with bacterial solution, and incubated at 37°C overnight; (5) Pick 4 single colonies for colony PCR (see Figure 3 ② in the above), the primers used are shown in SEQ ID NO.5 and SEQ ID NO.6 in Table 1, and 4 positive bacteria are randomly selected according to the results and cultured in a single tube with 4 mL LB liquid at 37°C in a shaking incubator overnight; (6) Extract the plasmid from the bacterial culture and send it for sequencing. The sequencing primers are SEQ ID NO.7~16 in Table 1. According to the sequencing results, the successfully connected and recombined colonies and lentiviral plasmid pLVX-IRES-Puro-dCas9-GCN4s are obtained. The nucleotide sequence of pLVX-IRES-Puro-dCas9-GCN4s is shown in SEQ ID NO.38 and Figure 8~Figure 10 .

[0043] 2. Construction of pLVX-IRES-Neo-Anti-GCN4-scFv-GFP-TET1CD plasmid (1) Using 82559 as a template, a product XA0139130-2A was obtained by PCR reaction. The primer sequences used are shown in SEQ ID NO. 17 and SEQ ID NO. 20 in Table 1, the PCR system is shown in sequence number 4 in Table 2, and the PCR reaction conditions are shown in Table 3; (2) Using 82559 as a template, a second product XA0139130-2B was obtained by PCR reaction. The primer sequences used are shown in SEQ ID NO. 18 and SEQ ID NO. 19 in Table 1, the PCR system is shown in sequence number 5 in Table 2, and the PCR reaction conditions are shown in Table 3; (3) Using XA0139130-2A and XA0139130-2B as templates, target fragment 2 was obtained by PCR reaction. The primer sequences used are shown in SEQ ID NO. 19 and SEQ ID NO. 20 in Table 1, the PCR system is shown in sequence number 6 in Table 2, and the PCR reaction conditions are shown in Table 3; (4) Gel recovery of target fragment 2, Anti-GCN4-scFv-GFP-TET1CD fragment (see Figure 3 ③), and at the same time, treat the PLVX-IRES-NEO plasmid with XHOI-NOTI, perform gel recovery, and obtain the linearized PLVX-IRES-NEO-XHOI-NOTI vector; 4 μL of target fragment 2, 3.5 μL of linearized PLVX-IRES-NEO-XHOI-NOTI vector, and 2.5 μL of recombinase were mixed, placed in a 50°C water bath for 25 min, placed for 3 min to cool down, and then transformed and coated with bacterial solution, and incubated at 37°C overnight; (5) Pick 4 single colonies for colony PCR (see Figure 3 ④ in the above), the primers used are shown in SEQ ID NO.21-22 in Table 1, and 4 positive bacteria are randomly selected according to the results and cultured in a single tube with 4 mL LB liquid at 37°C in a shaking incubator overnight; (6) Extract the plasmid from the bacterial culture and send it for sequencing. The sequencing primers are SEQ ID NO. 23 to 31 in Table 1. According to the sequencing results, the successfully connected and recombined colonies and the lentiviral plasmid pLVX-IRES-Neo-Anti-GCN4-scFv-GFP-TET1CD are obtained. The nucleotide sequence of pLVX-IRES-Neo-Anti-GCN4-scFv-GFP-TET1CD is shown in SEQ ID NO. 39 and Figure 11~Figure 13 .

[0044] 3. Lentivirus packaging and infection The connected lentiviral plasmids were introduced into human embryonic kidney 293T cells, two corresponding lentiviruses were packaged, and these two lentiviruses were used to successively infect human lung adenocarcinoma cell line PC-9 and human glioma cell line U251 to establish double lentiviral infected stable transfected cell lines. The details are as follows:

[0045] (1) Take human embryonic kidney 293T cells in the logarithmic growth phase. When the cell confluence reaches 90%, discard the supernatant, wash once with PBS, add an appropriate amount of trypsin, digest at room temperature for 30 seconds, add an appropriate amount of DMEM containing 10% fetal bovine serum to terminate the digestion, blow and mix the attached 293T cells into a cell suspension, and centrifuge at 800r / min for 5min. After the centrifugation, inoculate the cells into two 6cm dishes, with about 5×10 cells per dish. 6 The cells were cultured in an incubator at 37°C and 5% carbon dioxide. (2) After 12 hours, the cell confluence in the 6 cm dish should be around 80%. Replace the medium in each dish with 1.5 mL of fresh DMEM containing 10% fetal bovine serum and place the dish back in the 37°C, 5% carbon dioxide incubator for later use. (3) Prepare Solution A: 1 mL serum-free DMEM + 40 μL Lipofectamine 2000 (Thermo Fisher Scientific Catalog No. 11668), mix gently, and let stand at room temperature for 5 min; (4) Prepare Solution B: 500 μL serum-free DMEM + (1 μg pPMD2G plasmid + 3 μg pSPAX2 plasmid + 4 μg pLVX-IRES-Pμro-dCas9-GCN4s plasmid), mix gently; (5) Prepare Solution C: 500 μL serum-free DMEM + (1 μg pPMD2G plasmid + 3 μg pSPAX2 plasmid + 4 μg pLVX-IRES-Neo-Anti-GCN4-scFv-GFP-TET1CD plasmid), mix gently; (6) Add 1 / 2 of solution A to solution B to obtain AB working solution, and add 1 / 2 of solution A to solution C to obtain AC working solution. Mix gently and incubate at room temperature for 20 min. After the incubation is completed, remove the 6 cm dish from the incubator, add AB working solution dropwise to one dish, add AC working solution dropwise to the other dish, and return them to the cell incubator for culture. (7) After 6 hours, remove the transfection solution, add 5 mL of DMEM containing 10% fetal bovine serum to each dish, and continue to culture for 24 hours; after 24 hours, collect the supernatant of the two dishes, store at 4°C, add 5 mL of DMEM containing 10% fetal bovine serum again, and continue to culture for 48 hours; after 48 hours, mix the 293T cells in the two dishes into a cell suspension by pipetting, add them to the supernatant obtained from each dish for 24 hours, centrifuge at 2500 r / min for 5 minutes, and filter the obtained supernatant with a 0.45 μm filter. The obtained solutions are two lentivirus solutions: lentivirus-pLVX-IRES-Puro-dCas9-GCN4s and lentivirus-pLVX-IRES-Neo-Anti-GCN4-scFv-GFP-TET1CD; (8) PC-9 cells in the logarithmic growth phase were digested and centrifuged as described above and then inoculated into 6-well plates, with approximately 1.5 × 10 cells per well. 6 After culturing in a 37°C, 5% carbon dioxide incubator for 12 hours, the cell confluence should reach about 70%; (9) Take 1 mL of the aforementioned lentivirus-pLVX-IRES-Puro-dCas9-GCN4s virus solution, add 1 mL of DMEM containing 10% fetal bovine serum and 16 μg of Polybrene, and mix gently to obtain a virus infection solution containing 8 μg / mL Polybrene; remove the PC-9 6-well plate, discard the supernatant, add the virus infection solution to the wells, return to the incubator and continue culturing for 24 hours; after 24 hours, replace the medium with 2 mL of fresh DMEM containing 10% fetal bovine serum and continue culturing for 72 hours; after 72 hours, digest the cells according to the aforementioned method, centrifuge and discard the supernatant, add 5 mL of DMEM containing 1.5 μg / mL puromycin and 10% fetal bovine serum to the cell pellet, mix by pipetting to form a cell suspension, and inoculate in a 6 cm dish; (10) After 48 hours, the puromycin selection was completed, and the obtained PC-9 cells were a cell line stably transfected with dCas9-GCN4s. The cell line was digested, centrifuged, and resuspended according to the above method, and then inoculated into a 6-well plate. 1 mL of the above lentivirus-pLVX-IRES-Neo-Anti-GCN4-scFv-GFP-TET1CD virus solution was taken and infected again according to the same method. 72 hours after the infection, the cells were digested, centrifuged, and the cell pellet was resuspended in 5 mL of DMEM containing 2 mg / mL G418 geneticin and 10% fetal bovine serum, and inoculated into a 6 cm dish for selection and culture for 7 days. The obtained cells were the PC9 double lentivirus stable transfection line stably transfected with dCas9-GCN4s and Anti-GCN4-scFv-GFP-TET1CD (hereinafter referred to as PC-9 dCas9&TET1CD ); (11) According to the same method as (8)-(10), the U251 stable rotation system (hereinafter referred to as U251) is established. dCas9&TET1CD ).

[0046] 4. Exogenous introduction of sgRNA To PC-9 dCas9&TET1CD 、U251 dCas9&TET1CD sgRNA01 targeting the highly methylated region (synthesized by Nanjing GenScript Biotechnology Co., Ltd., where the targeting sequence is shown in SEQ ID NO.32 in Table 1) was transfected in the following steps: (1) Take PC-9 and PC-9 in the logarithmic growth phase dCas9&TET1CD Inoculate in 2 wells of a 6-well plate, approximately 1.5 × 10 6 After culturing for 12 hours in a 37°C, 5% carbon dioxide incubator, the cell confluence should reach about 70%. At this time, take out the 6-well plate and replace the medium in each well with 1.5 mL of DMEM containing 10% fetal bovine serum. (2) Prepare Solution A: 500 μL serum-free DMEM + 10 μL Lipofectamine 2000 (Thermo Fisher Scientific Catalog No. 11668), mix gently, and let stand at room temperature for 5 min; (3) Prepare Solution B: 500 μL serum-free DMEM + 5 μL sgRNA01 (20 μM, sequence see Table 1), mix gently; (4) Add solution A to solution B and mix gently to obtain the working solution. After standing at room temperature for 20 minutes, add the solution to PC-9 and PC-9. dCas9 &TET1CD 500 μL of working solution was added to each well and the cells were returned to the incubator for 6 h. After 6 h, each well was replaced with 2 mL of DMEM containing 10% fetal bovine serum and cultured for 72 h. (5) Follow the same method as (1) to (4) to add U251 and U251 dCas9&TET1CD sgRNA targeting the hypermethylated region was introduced into the

[0047] 5. Detection of DNA methylation levels in targeted regions to confirm the effectiveness of the system The genomic DNA of the treatment group and the control group was extracted and subjected to pyrophosphate sequencing after sulfite conversion (pyrophosphate sequencing Q48 technical service of Shanghai Jelan Biotechnology Co., Ltd.). The sequences of the amplification primers and sequencing primers used are shown in Table 1 SEQ ID NO.33~35. The 5' end of SEQ ID NO.34 was connected to Biotin to detect the methylation ratio of highly methylated CpG sites in the target region. The details are as follows:

[0048] (1) After completing the previous operation steps, use the Tiangen Genome Extraction Kit (Cat. No. DP304) to extract genomic DNA from each group. The experimental steps are carried out according to its instructions; (2) The genomic DNA of each group was subjected to bisulfite conversion using the Qiagen Bisulfite Conversion Kit (QIAGEN, Cat. No. 59104). After the conversion, desalting and washing were performed. The experimental steps were carried out according to the instructions. (3) The transformed genomic DNA was sent to Shanghai Jelan Biotechnology Co., Ltd. for Q48 pyrophosphate sequencing. The amplification and sequencing primers are shown in the table.

[0049] The sequencing results were analyzed and compared, and it was found that this system can successfully erase part of the methylation level in the hypermethylated region (see Figure 4 , Figure 5 , Table 4, Table 5).

[0050] Table 4 PC-9 demethylation effect data Table 5 U251 demethylation effect data It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0051] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0052] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A dual lentiviral plasmid system for DNA directed demethylation, characterized in that: The dual lentiviral plasmid system includes a dCas9-GCN4s sequence and an Anti-GCN4-scFv-TET1 sequence; The dCas9-GCN4s sequence is shown in SEQ ID No.36; The Anti-GCN4-scFv-GFP-TET1CD sequence is shown in SEQ ID No.

37.

2. The dual lentiviral plasmid system according to claim 1, characterized in that: The dual lentiviral plasmid system also includes an sgRNA targeting a hypermethylated region.

3. The dual lentiviral plasmid system according to claim 2, characterized in that: The nucleotide sequence of the sgRNA targeting the hypermethylated region is shown in SEQ ID NO.

32.

4. The method for constructing the dual lentiviral plasmid system according to claim 1, characterized in that: The following steps are involved: Insert the dCas9-GCN4s sequence into the downstream of the CMV promoter of the pLVX-IRES-Puro lentiviral plasmid vector to obtain the lentiviral plasmid pLVX-IRES-Puro-dCas9-GCN4s; Insert the Anti-GCN4-scFv-GFP-TET1CD sequence into the downstream of the CMV promoter of the pLVX-IRES-Neo lentiviral plasmid vector to obtain the lentiviral plasmid pLVX-IRES-Neo-Anti-GCN4-scFv-GFP-TET1CD; The lentiviral plasmid pLVX-IRES-Puro-dCas9-GCN4s is packaged to obtain lentiviral solution 1, and the lentiviral plasmid pLVX-IRES-Neo-Anti-GCN4-scFv-GFP-TET1CD is packaged to obtain lentiviral solution 2. The lentiviral solution 1 and the lentiviral solution 2 are used to infect the same cell, and sgRNA targeting the highly methylated region is introduced to obtain the dual lentiviral plasmid system.

5. The construction method according to claim 4, characterized in that: The cells are human lung adenocarcinoma cells PC-9.

6. The construction method according to claim 4, characterized in that: The cells are human glioma cells U251.

7. The construction method according to claim 4, characterized in that: Human embryonic kidney 293T cells were used for packaging.

8. The construction method according to claim 4, characterized in that: The nucleotide sequence of the sgRNA targeting the hypermethylated region is shown in SEQ ID NO.

32.

9. Use of the dual lentiviral plasmid system according to claim 1 in the production of lentiviral plasmids for targeted DNA demethylation.

10. Use of the dual lentiviral plasmid system according to claim 1 in directed demethylation of eukaryotic cell DNA.

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