SgRNA sequence for editing recombinant cell telomere and application of sgRNA sequence in targeted anti-tumor

Abstract

The invention discloses an sgRNA sequence for editing and recombining cell telomeres and application of the sgRNA sequence in targeted anti-tumor, and belongs to the technical field of biology. The nucleotide sequence of the sgRNA is as shown in SEQ ID NO. 1 or SEQ ID NO. 2. Experiments prove that the sgRNA can specifically recognize a telomere DNA repetitive sequence, then tumor specific promoters such as TERT or AFP are utilized to prepare a virus, SpCas9 nuclease and the sgRNA sequence are guided into tumor cells, and the telomeres of the tumor cells are cut and recombined, so that targeted gene editing of the tumor cell telomeres is realized, and the target gene editing of the tumor cells is realized. The tumor cell telomere sequence shortening and chromosome protection function losing can be efficiently caused, and finally the tumor cell death is caused.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an sgRNA sequence for editing recombinant cell telomeres and its targeted anti-tumor application. Background Art

[0002] Telomeres are a small segment of DNA-protein complex located at the end of linear chromosomes in eukaryotic cells. They play an important role in maintaining the stability of chromosome ends, controlling the number of cell divisions and cell aging. As cells divide, the length of telomeres gradually shortens and eventually reaches a critical value. When this value is exhausted, cells will no longer be able to continue dividing, thus entering a state of aging or death. Therefore, telomeres play a vital role in regulating the life cycle of cells. Changes in telomere length are closely related to the occurrence and development of tumors. The telomere extension mechanism of tumor cells gives tumor cells the ability to proliferate indefinitely. The telomere extension mechanism of tumor cells mainly involves two pathways: the telomerase-dependent pathway and the alternative telomere extension mechanism (ALT). Telomerase is an RNA-dependent DNA polymerase that can synthesize telomere repeat sequences (TTAGGG) at the ends of chromosomes, thereby maintaining telomere length and enabling cells to maintain vigorous division and proliferation capabilities. About 85% to 90% of tumor cells maintain telomere length by activating telomerase. However, in the case of telomerase inactivation or deficiency, some tumor cells can maintain telomere length through other mechanisms, which are collectively referred to as alternative telomere extension mechanisms.

[0003] The TERT gene encodes telomerase reverse transcriptase, an enzyme that is highly activated in most cancer cells. Telomerase reverse transcriptase can extend telomeres, allowing cancer cells to escape the normal process of cell aging and death. The TERT promoter is an important region that regulates the expression of the TERT gene. In many cancer types, TERT promoter mutations lead to abnormal activation of the TERT gene, thereby promoting the unlimited proliferation of tumor cells. Gene therapy or oncolytic viruses regulated by the TERT promoter can specifically target cancer cells. For example, by constructing a gene expression vector driven by the TERT promoter, toxic genes or pro-apoptotic genes can be efficiently expressed in cancer cells, thereby killing tumor cells. The AFP gene encodes alpha-fetoprotein, which is usually highly expressed in the fetal period and extremely low in normal tissues in adulthood. However, in some hepatocellular carcinomas and embryonal tumors, the AFP gene is abnormally highly expressed again. The AFP promoter is an important region that regulates the expression of the AFP gene and is often activated in hepatocellular carcinoma. Elevated serum levels of AFP are an important diagnostic marker for hepatocellular carcinoma. Detection of AFP levels in serum can help early detection and monitoring of liver cancer. By utilizing the AFP promoter to drive specific therapeutic genes, targeted gene therapy can be achieved in tumor cells with high AFP expression. For example, constructing an oncolytic virus or toxic gene expression vector driven by the AFP promoter can be specifically and efficiently expressed in hepatocellular carcinoma, thereby killing tumor cells and reducing side effects on normal tissues. Studies have shown that slight oxidative damage to tumor cell telomeres can induce tumor cell death, suggesting that tumor cell telomeres are one of the potential therapeutic targets for tumors. Abnormal activation of TERT and AFP promoters in tumor cells provides a unique opportunity for targeted cancer diagnosis and treatment. By studying and applying the specific regulatory mechanisms of these promoters, more accurate and efficient cancer treatments can be developed.

[0004] CRISPR / Cas9 technology is currently a very commonly used gene editing tool. Depending on the application, various types of Cas nucleases have been developed, such as the commonly used SpCas9 and SaCas9. Although in cancer research, CRISPR / Cas9 can be used to knock out or repair tumor-specific genes in order to inhibit tumor growth, different types of Cas nucleases need to be paired with different sgRNAs, which are the key factors in determining the accuracy and efficiency of the treatment plan. Summary of the invention

[0005] The purpose of the present invention is to provide a sgRNA sequence for editing recombinant cell telomeres and its targeted anti-tumor application to solve the problems existing in the above-mentioned prior art. The sgRNA can specifically recognize telomeric DNA repeat sequences, bind to TERT or AFP tumor-specific promoters, and continuously cut them using SpCas9 nuclease, thereby shortening the telomere length of tumor cells and ultimately killing tumor cells.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides an sgRNA for editing recombinant cell telomeres, wherein the nucleotide sequence of the sgRNA is shown as SEQ ID NO.1 or SEQ ID NO.2.

[0008] The present invention also provides a DNA molecule for transcribing the sgRNA.

[0009] The present invention also provides an expression cassette containing the sgRNA or the DNA molecule.

[0010] The invention also provides a recombinant vector containing the expression cassette.

[0011] The present invention also provides a recombinant bacterium containing the recombinant vector.

[0012] The present invention also provides a use of the sgRNA or the DNA molecule or the expression cassette or the recombinant vector or the recombinant bacteria in any of the following:

[0013] (1) Application in the preparation of viruses and drugs for shortening the telomere length of tumor cells;

[0014] (2) Application in the preparation of anti-tumor viruses and drugs.

[0015] Furthermore, the tumors include glioma, lung cancer, liver cancer and breast cancer.

[0016] Furthermore, when the promoter of the recombinant vector is CMV or TERT, the tumor includes glioma, lung cancer, liver cancer and breast cancer; when the promoter of the recombinant vector is AFP, the tumor is liver cancer.

[0017] The present invention also provides a drug for shortening tumor cell telomere length or anti-tumor, comprising the sgRNA.

[0018] The present invention discloses the following technical effects:

[0019] The sgRNA disclosed in the present invention can specifically recognize telomeric DNA repeat sequences, and then use the TERT or AFP tumor-specific promoter to guide the SpCas9 nuclease to the target gene fragment in the tumor cell, and continuously cut it, thereby shortening the telomere length of the tumor cell, and ultimately killing the tumor cell. Experiments have confirmed that 9 days after the telomere-cutting adenovirus infected liver cancer cells, breast cancer cells, lung cancer cells or glioma cells, the number of cells was significantly reduced and the length of the cell telomeres was significantly shortened. This shows that the present invention is based on CRISPR / Cas9 gene editing technology, and through the designed sgRNA and TERT or AFP tumor-specific promoter, targeted gene editing of tumor cell telomeres can be achieved, which can efficiently cause the shortening of the telomere sequence of tumor cells, and ultimately lead to the death of tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 is the pU6-(BsaI)_CBh-SpCas9 plasmid map;

[0022] Figure 2 is the sequencing result of sgRNA of the present invention;

[0023] Figure 3 This is a fluorescence microscopic observation of LN229 cells infected with CMV promoter telomere-cutting virus of the present invention;

[0024] Figure 4 This is a statistical diagram of the infection efficiency of LN229 cells infected with CMV promoter telomere-cutting virus of the present invention;

[0025] Figure 5 This is a comparison diagram of cell telomere length after LN229 cells were infected with CMV promoter telomere cutting virus of the present invention;

[0026] Figure 6 The figure shows the number of cells infected by the CMV promoter telomere-cutting virus in glioma cells (LN229), lung cancer cells (A546), liver cancer cells (HepG2) and breast cancer cells (MDA-MB-231) of the present invention, with a scale bar of 100 μm;

[0027] Figure 7The statistical graph of the cell count of glioma cells (LN229), lung cancer cells (A546), liver cancer cells (HepG2) and breast cancer cells (MDA-MB-231) infected with CMV promoter telomere cleavage virus of the present invention;

[0028] Figure 8 This is a fluorescence microscopic observation of LN229 cells infected with TERT promoter telomere-cutting virus of the present invention;

[0029] Fig. 9 This is a statistical diagram of the infection efficiency of LN229 cells infected with TERT promoter telomere-cutting virus of the present invention;

[0030] Fig.10 This is a comparison diagram of cell telomere length after LN229 cells were infected with TERT promoter telomere cutting virus of the present invention;

[0031] Fig.11 The figure is a statistical graph of infected cell counts of glioma cells (LN229), lung cancer cells (A546), liver cancer cells (HepG2) and breast cancer cells (MDA-MB-231) infected with TERT promoter telomere cutting virus of the present invention;

[0032] Fig.12 The fluorescence microscopic observation diagram of HepG2 cells and 293T cells infected with AFP promoter telomere cleavage virus of the present invention;

[0033] Fig.13 It is a statistical diagram of the infection efficiency of HepG2 cells and 293T cells infected with AFP promoter telomere cutting virus of the present invention;

[0034] Fig.14 This is a comparison diagram of cell telomere lengths after HepG2 cells and 293T cells of the present invention were infected with AFP promoter telomere-cutting virus;

[0035] Fig.15 It is a statistical graph of the infected cell count of the AFP promoter telomere-cutting virus in the glioma cells (LN229), lung cancer cells (A546), liver cancer cells (HepG2) and breast cancer cells (MDA-MB-231 infection) of the present invention. DETAILED DESCRIPTION

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0038] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0039] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0040] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0041] Example

[0042] 1. sgRNA design and plasmid construction

[0043] 1.1 Design of sgRNA

[0044] The present invention designs two sgRNAs for the repetitive sequence TTAGGG of human chromosome telomeric DNA, and an sgRNA (ng-sgRNA) that does not target any genomic sequence, and the corresponding sgRNA sequence information is shown in Table 1.

[0045] Table 1 sgRNA sequence information

[0046]

[0047] 1.2 Promoter design

[0048] The present invention designs three different promoters for SpCas9 protein, namely CMV promoter, TERT promoter and AFP promoter. Among them, CMV promoter is a promoter that comes with the plasmid, and the sequence information of TERT promoter and AFP promoter is obtained from the National Center for Biotechnology Information (NCBI) of the United States. The specific sequence information is shown in Table 2.

[0049] Table 2 Promoter sequence information

[0050]

[0051] 1.3 Plasmid construction

[0052] 1.3.1 Vector digestion

[0053] According to Table 3, prepare 50 μL of enzyme digestion system. Add various reagents in order according to the list, mix them by gently blowing with a pipette, centrifuge briefly, and place at 37°C for 3 hours or overnight. Perform agarose gel electrophoresis on the digestion product of the backbone plasmid pU6-(BsaI)_CBh-Cas9 (addgene, #135010) to recover the target band. The pU6-(BsaI)_CBh-Cas9 plasmid map is as follows Figure 1 As shown in the figure, regarding the TERT promoter and the AFP promoter, the promoters of the backbone plasmid pU6-(BsaI)_CBh-Cas9 were first replaced to obtain the backbone plasmids containing the TERT promoter and the AFP promoter, respectively.

[0054] Table 3 Enzyme digestion system

[0055]

[0056] 1.3.2 Primer annealing

[0057] The annealing system was prepared according to Table 4 (taking tel-sgRNA1 as an example), incubated in a 90°C water bath for 15 min, and naturally cooled to room temperature to obtain the annealing product, wherein the primer sequence information corresponding to each sgRNA is shown in Table 5.

[0058] Table 4 Annealing system

[0059]

[0060] Table 5 Primer sequences

[0061]

[0062]

[0063] 1.3.3 Ligation of annealed product and carrier

[0064] The ligation system was prepared according to Table 6 (taking tel-sgRNA1 as an example), and the double-digested linearized vector and the annealed double-stranded DNA were ligated by T4 DNA ligase, and the ligation was carried out at 16°C for 1-3 hours, or overnight, to obtain recombinant plasmids pU6-tel-sgRNA1-pCM V-SpCas9-EGFP, pU6-tel-sgRNA2-pCMV-SpCas9-EGFP, pU6-ng-sgRNA-pCMV-SpCas9-EGF P, pU6-tel-sgRNA1-pTERT-SpCas9-mCherry, pU6-tel-sgRNA2-pTERT-SpCas9-mCherry, pU6-ng-sgRNA-pTERT-SpCas9-mCherry, pU6-tel-sgRNA1-pAFP-SpCas9-EGFP, and pU6-tel-sgRNA2-p AFP-SpCas9-EGFP and pU6-ng-sgRNA-pAFP-SpCas9-EGFP.

[0065] Table 6 Connection system

[0066]

[0067] 1.3.4 Conversion

[0068] Add 10 μL of the ligation reaction product to 100 μL of competent cells, flick the tube wall several times to mix, and place on ice for 30 minutes. Heat shock at 42°C for 90 seconds, and incubate in an ice-water bath for 2 minutes. Add 500 μL of LB medium and place on a shaker at 37°C for 1 hour. Take an appropriate amount of bacterial solution and evenly spread it on a plate containing the corresponding antibiotics, and invert and culture it in a constant temperature incubator for 12-16 hours.

[0069] 1.3.5 Sequencing

[0070] The identified positive clone transformants were inoculated into an appropriate amount of LB liquid culture medium containing the corresponding antibiotics and cultured at 37°C for 12-16 hours. An appropriate amount of bacterial solution was taken for sequencing. The bacterial solution sequencing results of tel-sgRNA1, tel-sgRNA2 and ng-sgRNA are shown in Figure 2 As shown, the results were consistent with the expected insertion site.

[0071] 1.3.6 Plasmid extraction

[0072] The correctly sequenced bacterial solution was transferred to 50 mL of LB liquid culture medium containing the corresponding antibiotics, cultured at 37°C overnight, and plasmids were extracted using the AmMag Quatro Plasmid Purification Kit-24prep (water elution) kit. The qualified plasmids were extracted and entered the downstream process.

[0073] 2. Adenovirus packaging and quality testing

[0074] 2.1 Plasmid transfection and adenovirus harvest

[0075] (1) 24 hours before transfection, HEK293 cells in the logarithmic growth phase were digested with 0.25% trypsin, the cell density was adjusted to 30%-40% with DMEM medium containing 10% FBS, and the cells were re-seeded in a cell culture flask and cultured in a 37°C, 5% CO2 incubator. After about 24 hours, when the cell density reaches 50%-60%, the cells can be used for transfection;

[0076] (2) The medium was changed to serum-free medium 2 h before transfection;

[0077] (3) Mix the DNA solution (5 μg of shuttle plasmid and 5 μg of auxiliary plasmid) with DMEM to adjust the total volume to 50 μL and incubate at room temperature for 5 min; mix 10 μL of Lipofectamine 2000 reagent with 50 μL of DMEM and incubate at room temperature for 5 min; gently mix the diluted DNA solution with Lipofectamine 2000 without shaking and incubate at room temperature for 20 min to form a DNA / Lipofectamine 2000 transfection complex;

[0078] (4) Slowly add the transfection complex solution to the HEK293 cell culture medium, mix well, and culture in a 37°C, 5% CO2 cell culture incubator;

[0079] (5) After 6 h of incubation, discard the culture medium containing the transfection mixture, add 2 mL of PBS to wash once, shake gently to wash the remaining transfection mixture and then discard;

[0080] (6) Slowly add 5 mL of cell culture medium containing 10% serum and continue culturing in a 37°C, 5% CO2 incubator. Observe the growth of cells after transfection every day. If the culture medium turns yellow, add an appropriate amount of fresh complete culture medium as appropriate.

[0081] (7) About 10-15 days after transfection, observe HEK293 cells under a microscope to see if they begin to fall and show cytopathic effect (CPE);

[0082] (8) When most cells showed typical CPE and 50% of the cells were detached from the cell wall, the cells were collected by low-speed centrifugation and resuspended in 2 mL of OMEM. The cells were repeatedly frozen and thawed at -70°C / 37°C and shaken three times. The cells were centrifuged at 4°C and 7000 g for 5 min, and the viral supernatant was collected and stored at -70°C.

[0083] 2.2 Adenovirus amplification and purification

[0084] 2.2.1 First round of amplification and purification

[0085] (1) HEK293 cells in good growth condition were transferred into a 10 cm cell culture dish. When the cell confluence reached 60%, the old culture medium was discarded, and an appropriate amount of crude extract of replication-deficient adenovirus after successful recombinant was added. The cells were placed in a cell culture incubator and incubated for 90 min. After that, 10 mL of complete culture medium was added and the culture was continued;

[0086] (2) When most cells showed typical CPE and 50% of the cells were detached from the cell wall, the cells were collected by low-speed centrifugation and resuspended in 2 mL of DMEM, and repeatedly frozen and thawed and shaken in liquid nitrogen / 37°C for 3 times;

[0087] (3) Centrifuge at 5000 g for 5 min at 4°C, collect the viral supernatant and store it at -70°C.

[0088] 2.2.2 Second round of amplification and purification

[0089] (1) HEK293 cells in good growth condition were transferred into a 10 cm cell culture dish. When the cell confluence reached 90%, the old culture medium was discarded, and an appropriate amount of the virus solution obtained from the first round of amplification was added. The dish was placed in a cell culture incubator for 90 min, and then 10 mL of complete culture medium was added and cultured continuously;

[0090] (2) When most cells showed typical CPE and 50% of the cells were detached from the cell wall, the cells were collected by low-speed centrifugation and resuspended in 10 mL of DMEM, and repeatedly frozen and thawed and shaken in liquid nitrogen / 37°C for 3 times;

[0091] (3) Centrifuge at 5000 g for 5 min at 4°C and collect the supernatant;

[0092] (4) Transfer the supernatant to a 1.5 mL centrifuge tube and centrifuge at 4°C and 12,000 rpm for 10 min to fully remove cell debris;

[0093] (5) Filter the supernatant with a 0.22 μm filter into a 20 mL ultrafiltration concentration tube;

[0094] (6) Centrifuge at 4°C and 5000 g, adjusting the centrifugation time to concentrate the supernatant to the desired volume;

[0095] (7) The concentrated virus solution is filtered for a second time, packaged as required, and stored at -70°C;

[0096] (8) Prepare samples for testing.

[0097] 2.3 Adenovirus quality testing

[0098] 2.3.1 Physical index detection

[0099] (1) pH value determination: The pH value of adenovirus storage solution ranges from 6.4 to 7.0;

[0100] (2) Determination of viscosity: Use a 20-200 μL pipette to slowly aspirate 50 μL of adenovirus storage liquid. No obvious viscosity or aspiration lag should be observed.

[0101] 2.3.2 Sterility testing

[0102] The virus was added to HEK293 cells for verification. After 24 hours of normal culture, microscopic examination showed no bacterial or fungal contamination. At the same time, referring to the empty cell group, there were no obvious particles in the intercellular space and the culture medium was clear and transparent.

[0103] 2.3.3 Adenovirus titer determination - endpoint dilution method

[0104] (1) 24 h before the experiment, a 96-well plate was plated and 100 μL of HEK293 cell suspension (containing approximately 1×10 3 cells;

[0105] (2) Prepare 12 sterile EP tubes. Add 990 μL of complete culture medium to the first EP tube and 900 μL of complete culture medium to each of the remaining 11 EP tubes.

[0106] (3) Dilution of the virus solution to be tested: Take 10 μL of the adenovirus stock solution and add it to the first EP tube for a 1:100 dilution; then take this as the starting point, take 100 μL of the virus dilution solution and add it to the next EP tube for a 1:10 dilution, until the dilution reaches 10 -13 ;

[0107] (4) Discard the old culture medium in the 96-well plate and add 10 -13 Up to 10 -6 Each well of the first 10 wells in each row was filled with 90 μL of virus dilution, and the 11th to 12th wells were filled with 90 μL of complete medium without virus as a control.

[0108] (5) Place the 96-well plate in a 37°C, 5% CO2 cell culture incubator and continue culturing;

[0109] (6) After 10 days, observe the cytopathic effect, count the number of cell wells showing CPE, calculate the CPE positive rate after treatment with virus solution of each dilution, and calculate the virus titer;

[0110] (7) Virus titer analysis (Spearman-Karber Method):

[0111] Virus titer = 10 (x+0.8) (PFU / mL)*,

[0112] Where X is 10 -1 to 10 -13 The sum of CPE positive rates at different dilutions,

[0113] *The conditions for using the formula are: A. The negative control showed no CPE and growth inhibition; B. All wells with the minimum dilution concentration of crude virus extract showed CPE.

[0114] 3. Cell infection

[0115] The present invention further infected the above adenovirus in glioma cells (LN229), lung cancer cells (A546), liver cancer cells (HepG2) and breast cancer cells (MDA-MB-231). The specific method of infection is: 1×10 5 The cells were inoculated into a 6 cm culture dish and cultured in a 37°C incubator for 16-24 hours until the cell confluence reached 30-40%. According to the virus titer, the corresponding amount of virus was added to reach moi=100.

[0116] Calculation formula: moi = (virus titer × virus volume) / cell number.

[0117] The cells were cultured in a 37°C incubator for 16 h, then replaced with complete culture medium and continued to be cultured. About 72 h after infection, the infection efficiency was observed.

[0118] For the telomere-cutting adenovirus of the CMV promoter, the infection efficiency of glioma cells (LN229) was as follows Figure 3 and Figure 4 As shown, Figure 3 This is a fluorescence microscopic observation image taken 72 hours after infection. Figure 4 The results show that the infection method of the present invention can achieve a better infection effect.

[0119] For the telomere-cutting adenovirus expressing the TERT promoter, the infection efficiency of glioma cells (LN229) was as follows Figure 8 and Fig. 9 As shown, Figure 8 This is a fluorescence microscopic observation image taken 72 hours after infection. Fig. 9 The results show that the infection method of the present invention can achieve a better infection effect.

[0120] For the telomere-cutting adenovirus expressing the AFP promoter, the infection efficiency of hepatoma cells (HepG2) and 293T cells was as follows Fig.12 and Fig.13 As shown, Fig.12 This is a fluorescence microscopic observation image taken 72 hours after infection. Fig.13The results show that for HepG2 cells, the infection method of the present invention can achieve a better infection effect, while since 293T cells do not contain AFP promoter, the infection efficiency of 293T cells is 0.

[0121] For the telomere-cutting adenovirus of the CMV promoter, the day of virus infection was recorded as day 0. The number of cells in each group was observed on the 9th day after virus infection of different tumor cells. Figure 6 As shown, the number of cells in different groups was counted on days 3, 6, and 9. Since the effect of infection with tel-sgRNA2 is similar to that of tel-sgRNA1, infection with tel-sgRNA1 is used as an example. The statistical results are shown in Figure 7 As shown, compared with the control virus group, the number of cells in the target virus group was significantly reduced, indicating that the virus can cut the telomeres of glioma cells (LN229), lung cancer cells (A546), liver cancer cells (HepG2), and breast cancer cells (MDA-MB-231), induce telomere shortening of different tumor cells, and ultimately kill various tumor cells.

[0122] For the telomere-cutting adenovirus of the TERT promoter, the day of virus infection was recorded as day 0, and the number of cells in different groups was counted on days 3, 6, and 9. Since the effect of infection with TERT-tel-sgRNA2 was similar to that of TERT-tel-sgRNA1, infection with TERT-tel-sgRNA1 was used as an example. The statistical results are shown in Fig.11 As shown, compared with the control virus group, the number of cells in the target virus group was significantly reduced, indicating that the virus can cut the telomeres of glioma cells (LN229), lung cancer cells (A546), liver cancer cells (HepG2), and breast cancer cells (MDA-MB-231), induce telomere shortening of different tumor cells, and ultimately kill various tumor cells.

[0123] For the telomere-cutting adenovirus of the AFP promoter, the day of virus infection was recorded as day 0, and the number of cells in different groups was counted on days 3, 6, and 9. Since the effect of infection with AFP-tel-sgRNA2 was similar to that of AFP-tel-sgRNA1, infection with AFP-tel-sgRNA1 was used as an example. The statistical results are shown in Fig.15 As shown, compared with the control virus group, the number of cells in the HepG2 cell infection target virus group was significantly reduced, indicating that the virus can cut the telomeres of liver cancer cells (HepG2), while for glioma cells (LN229), lung cancer cells (A546), and breast cancer cells (MDA-MB-231) that do not contain the AFP promoter, the telomere-cutting adenovirus of the AFP promoter has no obvious effect, indicating that the telomere-cutting adenovirus of the AFP promoter can target and kill liver cancer cells.

[0124] 4. Detection of cell telomere length after viral infection

[0125] After infection, cells with higher infection efficiency were selected, digested with 0.05% trypsin, and the cell suspension was transferred to a 1.5 mL EP tube. The genome was extracted using a genome extraction kit (TIANGEN), and the telomere length was detected using human telomere length quantitative qPCR analysis.

[0126] The specific detection method is as follows: prepare the reaction systems in Table 7 and Table 8, whose corresponding primer sequence information is shown in Table 9 and Table 10, and use a fluorescent quantitative PCR instrument for detection, and the reaction procedures are shown in Table 11 and Table 12.

[0127] Table 7 Sample DNA qPCR reaction system

[0128]

[0129] Table 8 Internal reference DNA qPCR reaction system

[0130]

[0131] Table 9 Sample telomere primer sequences

[0132]

[0133] Table 10 Internal reference gene primer sequences

[0134]

[0135] Table 11 Sample DNA qPCR reaction procedure

[0136]

[0137] Table 12 Internal reference DNA qPCR reaction procedure

[0138]

[0139] The qPCR results were exported for analysis, and a standard curve was generated using the Biosystems Prism 7700 Sequence Detection System, which was used to calculate the input amount for each sample.

[0140] Average telomere length ratio (ATLR) = relative input amount of sample DNA qPCR / relative input amount of internal reference DNA qPCR

[0141] For the telomere-cutting adenovirus of the CMV promoter, the results were as follows Figure 5 As shown, the results showed that 9 days after LN229 cells were infected with telomere-cutting adeno-associated virus, the length of cell telomeres was significantly shortened, indicating that the virus can induce a significant shortening of tumor cell telomere length.

[0142] For the telomere-cutting adenovirus expressing the TERT promoter, the results were as follows Fig.10 As shown, the results showed that 9 days after LN229 cells were infected with telomere-cutting adeno-associated virus, the length of cell telomeres was significantly shortened, indicating that the virus can induce a significant shortening of tumor cell telomere length.

[0143] For the telomere-cutting adenovirus expressing the AFP promoter, the results were as follows Fig.14 As shown, the results showed that 9 days after the telomere-cutting adeno-associated virus infected liver cancer cells (HepG2) and 293T cells, the telomere length of HepG2 cells was significantly shortened, while the telomere length of 293T cells did not change significantly, indicating that the telomere-cutting adenovirus of the AFP promoter can target and induce a significant shortening of the telomere length of HepG2 liver cancer cells.

[0144] In summary, the present invention successfully achieves effective cutting of telomere DNA repeat sequences by designing and constructing sgRNA that can target telomeric DNA repeat sequences, and uses CRISPR / Cas9 technology to achieve a large number of telomeric DNA double-strand breaks and recombination, thereby achieving the purpose of telomere shortening. Combined with the TERT or AFP tumor-specific promoter, the SpCas9 nuclease is guided to the target gene fragment in the tumor cell, which can achieve targeted telomere cutting of specific tumor cells. The present invention detects the length of the telomere after cutting, and the results show that the telomere length of the tumor cells is significantly shortened, and a large number of tumor cells die after the telomere is shortened, suggesting that the CRISPR / Cas9 gene editing method constructed by the sgRNA designed by the present invention can be used as a potential new tumor treatment method.

[0145] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A sgRNA for editing recombinant cell telomeres, characterized in that: The nucleotide sequence of the sgRNA is shown as SEQ ID NO.1 or SEQ ID NO.

2.

2. A DNA molecule that transcribes the sgRNA according to claim 1.

3. An expression cassette containing the sgRNA of claim 1 or the DNA molecule of claim 2.

4. A recombinant vector containing the expression cassette according to claim 3.

5. A recombinant bacterium containing the recombinant vector according to claim 4.

6. Use of the sgRNA according to claim 1, the DNA molecule according to claim 2, the expression cassette according to claim 3, the recombinant vector according to claim 4, or the recombinant bacterium according to claim 5 in any of the following: (1) Application in the preparation of viruses and drugs for shortening the telomere length of tumor cells; (2) Application in the preparation of anti-tumor viruses and drugs.

7. The use according to claim 6, characterized in that The tumors include glioma, lung cancer, liver cancer and breast cancer.

8. The use according to claim 7, characterized in that When the promoter of the recombinant vector is CMV or TERT, the tumor includes glioma, lung cancer, liver cancer and breast cancer; when the promoter of the recombinant vector is AFP, the tumor is liver cancer.

9. A virus and drug for shortening tumor cell telomere length or anti-tumor, characterized in that: Comprising the sgRNA of claim 1.

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