Reagents and uses to increase the level of rev7 promoter methylation

Abstract

This invention relates to reagents and uses for increasing REV7 promoter methylation levels, specifically providing the use of reagents that inhibit REV7 gene expression in the preparation of drugs for treating cancer, drugs for improving the efficacy of anticancer drugs, and drugs for increasing the sensitivity of tumor cells to anticancer drugs, wherein the anticancer drugs are chemotherapeutic drugs and / or PARP inhibitors. Inhibition of REV7 gene expression can significantly increase the sensitivity of tumors to platinum-based drugs and PARP inhibitors.

Description

Technical Field

[0001] This invention relates to epigenetics and tumor treatment, specifically to epigenetic editing techniques that inactivate REV7 to increase drug sensitivity in ovarian cancer. Background Technology

[0002] Platinum-based chemotherapy is the first-line drug for ovarian cancer in clinical practice. It induces interstrand cross-linking (ICL) of DNA, inhibiting transcription and replication, and promoting cell cycle arrest and apoptosis. Currently, the targeted drug PARPi is only used for maintenance therapy in patients sensitive to platinum-based chemotherapy or relapsed patients with BRCA1 / 2 mutations. Its efficacy is closely related to heart rate repair deficiency (HRD). Drug resistance that occurs during treatment is one of the major problems in the clinical treatment of ovarian cancer. Drug resistance may occur due to changes in genes and epigenetics, helping cancer cells adapt to drug effects through various pathways such as stress, DNA damage, and apoptosis. For a long time, there has been little progress in the treatment of ovarian cancer, and drug resistance remains one of the major problems in clinical ovarian cancer treatment. Developing sensitization methods is of great significance.

[0003] Epigenetic editing systems selectively knock down drug resistance-related genes, causing platinum-based chemotherapy drugs / PARPi inhibitors to kill cells. Therefore, studying the epigenetic regulatory mechanisms of drug sensitivity in ovarian cancer can provide new intervention targets for drug sensitization and has important clinical translational significance. Summary of the Invention

[0004] To address the aforementioned deficiencies, the first aspect of this invention provides the use of a reagent for inhibiting REV7 gene expression in the preparation of drugs for treating cancer, drugs for improving the efficacy of anticancer drugs, and drugs for improving the sensitivity of tumor cells to anticancer drugs, wherein the anticancer drug is a chemotherapeutic drug and / or a PARP inhibitor.

[0005] In one or more embodiments, the agent for inhibiting REV7 gene expression is: (1) an agent that increases the methylation level of the REV7 promoter region or a fragment thereof; or (2) an inhibitory molecule that specifically interferes with the transcription and / or expression of the REV7 gene.

[0006] In one or more embodiments, the REV7 promoter region is the region from 2000 bp upstream to 1000 bp downstream of the transcription start site.

[0007] In one or more embodiments, the REV7 promoter region is from position 11753707 to position 11750708 of human chromosome 1, and the segment of the REV7 promoter region is a segment from position 11753707 to position 11750708 of human chromosome 1 containing positions 11751554 to 11751655.

[0008] In one or more embodiments, the REV7 promoter region is from position 11753707 to position 11750708 of human chromosome 1, and the segment of the REV7 promoter region is a segment from position 11752480 to position 11752544 of human chromosome 1 from position 11753707 to position 11750708.

[0009] In one or more embodiments, the REV7 promoter region is as shown in SEQ ID NO:20. In one or more embodiments, increasing the methylation level of the REV7 promoter region or a fragment thereof refers to increasing the average methylation level of any one or all of the CG dinucleotides in the sequence from positions 11751554 to 11751655 of human chromosome 1.

[0010] In one or more embodiments, increasing the methylation level of the REV7 promoter region or a fragment thereof means increasing the average methylation level of any one or all of the CG dinucleotides in the sequence from position 11752480 to position 11752544 of human chromosome 1.

[0011] In one or more embodiments, the reagent that increases the methylation level of the REV7 promoter region or a fragment thereof comprises: (i) sgRNA or its complementary sequence, or (ii) a nucleic acid construct containing said sgRNA or its complementary sequence.

[0012] In one or more embodiments, the nucleic acid construct is the sgRNA or its complementary sequence, or a cloning vector or expression vector.

[0013] In one or more embodiments, the sgRNA has a sequence as shown in SEQ ID NO:1, 2 and / or SEQ ID NO:3 or a variant thereof having at least 80% sequence identity.

[0014] In one or more embodiments, the reagent further comprises an epigenetic editing reagent.

[0015] In one or more embodiments, the epigenetic editing reagent comprises a DNA methyltransferase or its expression vector, or a Cas enzyme or its expression vector.

[0016] In one or more embodiments, the DNA methyltransferase is selected from one or more of DNMT1, DNMT2, DNMT3A, or DNMT3B; the DNA methyltransferase optionally further comprises a DNA-binding protein, such as DNMT3L. Preferably, the N-segment or C-segment of the DNA methyltransferase is fused with DNMT3L.

[0017] In one or more embodiments, the Cas enzyme is Cas9, preferably a Cas9 mutant dCas9 without endonuclease activity. Preferably, the N-segment or C-segment of the Cas enzyme is fused with a KRAB domain.

[0018] In one or more embodiments, the epigenetic editing reagent further comprises a marker or expression vector thereof for detection, such as a peptide epitope or a fluorescent protein.

[0019] In one or more embodiments, the epigenetic editing reagent comprises a fusion protein or its expression vector, the fusion protein comprising...

[0020] (1) DNA methyltransferases, such as DNMT1, DNMT2, DNMT3A, or DNMT3B, or

[0021] (2) Cas enzyme, such as Cas9, preferably the Cas9 mutant dCas9 which lacks endonuclease activity.

[0022] Optionally, it may also contain DNA-binding proteins located in the N- or C-segment of DNA methyltransferases, such as DNMT3L.

[0023] Optionally, it may also include a KRAB domain located in the N or C segment of the Cas enzyme.

[0024] The optional component also includes nuclear positioning signals.

[0025] Optional features may also include biomarkers for detection, such as peptide epitopes and fluorescent proteins.

[0026] In one or more embodiments, the fusion protein has a connector between its domains or components, such as XTEN80.

[0027] In one or more embodiments, the fusion protein comprises, from the N-terminus to the C-terminus, the following:

[0028] dCas9, KRAB structural domains

[0029] dCas9, DNMT3A, DNMT3L,

[0030] KRAB structural domain, dCas9, DNMT3A, DNMT3L, or

[0031] dCas9, DNMT3A, DNMT3L, KRAB structural domains.

[0032] In one or more embodiments, (2) the repressor molecule targets the REV7 gene or its transcript. In one or more embodiments, the repressor molecule is selected from the group consisting of (a) small molecule compounds, antisense nucleic acids, microRNAs, siRNAs, RNAi, dsRNAs, sgRNAs, antibodies, or combinations thereof, and (b) nucleic acid constructs that can express or form (a).

[0033] In one or more embodiments, the inhibitory molecule has any of the sequences shown in SEQ ID NO:8-13.

[0034] In one or more embodiments, the inhibitory molecule has the sequences shown in (i) SEQ ID NO:8 and 9, or (ii) SEQ ID NO:10 and 11, or (iii) SEQ ID NO:12 and 13. Preferably, the inhibitory molecule has the sequences shown in SEQ ID NO:8 and 9.

[0035] In one or more embodiments, the inhibitor further comprises a Cas enzyme (e.g., Cas9), its coding sequence, and / or a nucleic acid construct expressing the Cas enzyme.

[0036] In one or more embodiments, the cancer is a cancer with upregulated REV7 expression, and the tumor cells are tumor cells with upregulated REV7 expression. In one or more embodiments, the cancer is not a cancer with downregulated REV7 expression, and the tumor cells are not tumor cells with downregulated REV7 expression.

[0037] In one or more embodiments, the cancer is ovarian cancer, and the tumor cells are ovarian cancer cells. In one or more embodiments, the cancer is ovarian cancer with upregulated or no significant change in REV7 expression, and the tumor cells are ovarian cancer cells with upregulated or no significant change in REV7 expression.

[0038] In one or more embodiments, the tumor cells are SKOV3 cells, OVCAR3 cells, CoC1 cells, or A2780 cells. In one or more embodiments, the tumor cells are SKOV3 cells, OVCAR3 cells, CoC1 cells, or A2780 cells with upregulated or no significant change in REV7 expression.

[0039] In one or more embodiments, the chemotherapy drug is a platinum-based drug.

[0040] In one or more embodiments, the platinum-based drug includes cisplatin, carboplatin, nedaplatin, oxaliplatin, and lobaplatin, preferably cisplatin.

[0041] In one or more embodiments, the PARP inhibitor includes olaparib, niraparib, fluzoparib, pamiparib, and preferably, the PARP inhibitor is olaparib.

[0042] The present invention also provides the use of reagents for inhibiting REV7 gene expression and anticancer drugs in the preparation of medicaments for treating cancer, wherein the reagents for inhibiting REV7 gene expression, anticancer drugs, and cancers are as described in any embodiment of the first aspect of the present invention.

[0043] The present invention also provides a method for treating cancer, comprising administering a therapeutically effective amount of a reagent for inhibiting REV7 gene expression and a therapeutically effective amount of an anticancer drug, wherein the reagent for inhibiting REV7 gene expression, the anticancer drug, and the cancer are as described in any embodiment of the first aspect of the present invention.

[0044] In one or more embodiments, the anticancer drug is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after administration of a reagent that inhibits REV7 gene expression.

[0045] The present invention also provides a non-therapeutic method for reducing REV7 expression in cells in vitro, including increasing the methylation level of the REV7 promoter region or fragments thereof.

[0046] In one or more embodiments, the REV7 promoter region is the region from 2000 bp upstream to 1000 bp downstream of the transcription start site.

[0047] In one or more embodiments, the REV7 promoter region is from position 11753707 to position 11750708 of human chromosome 1, and the segment of the REV7 promoter region is a segment of position 11751554 to position 11751655 of human chromosome 1 from position 11753707 to position 11750708.

[0048] In one or more embodiments, the REV7 promoter region is from position 11753707 to position 11750708 of human chromosome 1, and the segment of the REV7 promoter region is a segment from position 11752480 to position 11752544 of human chromosome 1 from position 11753707 to position 11750708.

[0049] In one or more embodiments, the REV7 promoter subregion is shown as SEQ ID NO:20.

[0050] In one or more embodiments, increasing the methylation level of the REV7 promoter region or a fragment thereof means increasing the average methylation level of any one or all of the CG dinucleotides in the sequence from position 11751554 to position 11751655 of human chromosome 1.

[0051] In one or more embodiments, increasing the methylation level of the REV7 promoter region or a fragment thereof means increasing the average methylation level of any one or all of the CG dinucleotides in the sequence from position 11752480 to position 11752544 of human chromosome 1.

[0052] In one or more embodiments, the method includes:

[0053] Introduce the following reagents into cells: (1) sgRNA or its complementary sequence, or a nucleic acid construct containing said sgRNA or its complementary sequence, and (2) an epigenetic editing reagent; or

[0054] Introduce the following reagents into cells containing epigenetic editing reagents: sgRNA or its complementary sequence, or a nucleic acid construct containing said sgRNA or its complementary sequence.

[0055] The epigenetic reagent is as described in any embodiment of the first aspect of this document.

[0056] The present invention also provides a kit or composition comprising (1) sgRNA or its complementary sequence, or a nucleic acid construct containing said sgRNA or its complementary sequence, and (2) an epigenetic editing reagent.

[0057] In one or more embodiments, the nucleic acid construct is the sgRNA or its complementary sequence, or a cloning vector or expression vector.

[0058] In one or more embodiments, the sgRNA has a sequence as shown in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, or a variant thereof having at least 80% sequence identity.

[0059] In one or more embodiments, the epigenetic editing reagent comprises a DNA methyltransferase or its expression vector, or a Cas enzyme or its expression vector.

[0060] In one or more embodiments, the DNA methyltransferase is selected from one or more of DNMT1, DNMT2, DNMT3A, or DNMT3B; the DNA methyltransferase optionally further comprises a DNA-binding protein, such as DNMT3L. Preferably, the N-segment or C-segment of the DNA methyltransferase is fused with DNMT3L.

[0061] In one or more embodiments, the Cas enzyme is Cas9, preferably a Cas9 mutant dCas9 without endonuclease activity. Preferably, the N-segment or C-segment of the Cas enzyme is fused with a KRAB domain.

[0062] In one or more embodiments, the epigenetic editing reagent further comprises a marker or expression vector thereof for detection, such as a peptide epitope or a fluorescent protein.

[0063] In one or more embodiments, the epigenetic editing reagent comprises a fusion protein or its expression vector, the fusion protein comprising...

[0064] (1) DNA methyltransferases, such as DNMT1, DNMT2, DNMT3A, or DNMT3B, or

[0065] (2) Cas enzyme, such as Cas9, preferably the Cas9 mutant dCas9 which lacks endonuclease activity.

[0066] Optionally, it may also contain DNA-binding proteins located in the N- or C-segment of DNA methyltransferases, such as DNMT3L.

[0067] Optionally, it may also include a KRAB domain located in the N or C segment of the Cas enzyme.

[0068] The optional component also includes nuclear positioning signals.

[0069] Optional features may also include biomarkers for detection, such as peptide epitopes and fluorescent proteins.

[0070] In one or more embodiments, the fusion protein has a connector between its domains or components, such as XTEN80.

[0071] In one or more embodiments, the fusion protein comprises, from the N-terminus to the C-terminus, the following:

[0072] dCas9, KRAB structural domains

[0073] dCas9, DNMT3A, DNMT3L,

[0074] KRAB structural domain, dCas9, DNMT3A, DNMT3L, or

[0075] dCas9, DNMT3A, DNMT3L, KRAB structural domains. Attached Figure Description

[0076] Figure 1 REV7 gene promoter methylation levels in different ovarian tissue / cell samples. The horizontal axis represents the various samples (normal ovarian tissue, ovarian cancer cells, ovarian cancer tissue), and n represents the number of samples; the vertical axis represents the average methylation level at 8 sites.

[0077] Figure 2: Methylation level of the REV7 gene promoter in primary / recurrent ovarian cancer tissues. The horizontal axis represents various samples (recurrent ovarian cancer tissues, primary ovarian cancer tissues), and n represents the number of samples; the vertical axis represents the methylation level.

[0078] Figure 3 Kaplan-Meier plot. Solid lines represent highly methylated samples, and dashed lines represent low-methylated samples. Survival rate is expressed in months post-surgery, and significance is defined as p<0.05.

[0079] Figure 4 The effect of REV7 promoter methylation status on homologous recombination defect (HRD). The horizontal axis represents chromosome location, and the vertical axis represents methylation level. Square icons represent REV7 promoter methylation in HRD-positive samples, and circular icons represent REV7 promoter methylation in HRD-negative samples.

[0080] Figure 5 Correlation between REV7 promoter methylation and HRD score. A. Correlation between the average methylation of 8 consecutive detection sites in the REV7 promoter and HRD score. B. Correlation between the methylation level of a single site in the REV7 promoter and HRD score. The x-axis represents HRD score, and the y-axis represents the REV7 promoter methylation level.

[0081] Figure 6 REV7 gene promoter methylation regulates gene expression. A. Azacytidine treatment affects REV7 gene expression in ovarian cancer cells SKOV3, A2780, and OVCAR3. The x-axis represents cell lines, and the y-axis represents gene expression levels. B. Dual-luciferase reporter gene assay detects the effect of REV7 promoter methylation on transcriptional activity. The x-axis represents the control reporter vector pCpGL-basic and the REV7 promoter unmethylated / methylated reporter vectors, respectively. The y-axis represents REV7 transcriptional activity. (*P<0.05, **p<0.01, ***p<0.001, ****p<0.0001)

[0082] Figure 7 : siRNA knockout of REV7 gene expression. The x-axis represents cell lines treated with different siRNAs, and the y-axis represents the change in REV7 gene expression relative to the control siRNA treatment. (*P<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0083] Figure 8REV7 gene knockdown and overexpression. A. REV7 gene knockdown. B. REV7 gene overexpression. The x-axis represents cell lines, and the y-axis represents the expression level of the REV7 gene in cells. (*P<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0084] Figure 9 Effect of REV7 gene knockdown on HR repair capacity in A2780 cells. A. Dual immunofluorescence staining of RAD51 / γH2AX in A2780 cells exposed to 0.75 μg / ml DDP or 10 μM Olaparib. Scale bar: 5 μm. B. Quantification of γH2AX and RAD51 by immunofluorescence focusing in up to 100 cells per group of DDP-treated A2780 cells. C. Quantification of γH2AX and RAD51 by immunofluorescence focusing in up to 100 cells per group of Olaparib-treated A2780 cells. γH2AX protein showed green fluorescence, indicating intracellular DNA damage, while RAD51 showed red fluorescence, indicating intracellular DNA repair. (*P<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0085] Figure 10 Effect of REV7 gene knockdown on HR repair capacity in SKOV3 cells. A. Dual immunofluorescence staining of RAD51 / γH2AX in SKOV3 cells exposed to 0.75 μg / ml DDP or 100 μM Olaparib. Scale bar: 5 μm. B. Quantification of γH2AX and RAD51 by immunofluorescence focusing in up to 100 cells per group of DDP-treated SKOV3 cells. C. Quantification of γH2AX and RAD51 by immunofluorescence focusing in up to 100 cells per group of Olaparib-treated SKOV3 cells. γH2AX protein showed green fluorescence, indicating intracellular DNA damage, while RAD51 showed red fluorescence, indicating intracellular DNA repair. (*P<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0086] Figure 11Effect of REV7 overexpression on HR repair capacity of SKOV3 cells. A. Dual immunofluorescence staining of RAD51 / γH2AX in SKOV3 cells exposed to 0.75 μg / ml DDP or 100 μM Olaparib. Scale bar: 5 μm. B. Quantification of γH2AX and RAD51 by immunofluorescence focusing in up to 100 cells per group of DDP-treated SKOV3 cells. C. Quantification of γH2AX and RAD51 by immunofluorescence focusing in up to 100 cells per group of Olaparib-treated SKOV3 cells. γH2AX protein is green fluorescent, indicating intracellular DNA damage; RAD51 is red fluorescent, indicating intracellular DNA repair. (*P<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0087] Figure 12 Effect of REV7 gene knockdown on drug sensitivity of OC cells. A. Proliferation curves of A2780 cells treated with 0.75 μg / ml DDP and 10 μM Olaparib for 4 days after REV7 gene knockdown, expressed as OD450. A. Proliferation curves of SKOV3 cells treated with 0.75 μg / ml DDP and 100 μM Olaparib for 4 days after REV7 gene knockdown, expressed as OD450. C. Bar plots show the OD450 of A2780 and SKOV3 cells after 4 days of drug treatment, expressed as mean ± SD. (*P<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0088] Figure 13 REV7 overexpression affects cell clonal capacity. A. 14 days after REV7 overexpression, the number of clones in three cell types (A2780, SKOV3, and OVCAR3) changed. B. Bar graphs show the changes in the number of clones in the three cell types. (*P<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0089] Figure 14Effects of REV7 siRNA knockdown and overexpression on A2780 cell migration. A. Scratch width of A2780 cells at day 0, day 2, and day 4 after 24 h of REV7 gene knockdown and overexpression plasmid transfection. B. Migration distance of A2780 cells in the REV siRNA group and control group at day 2 and day 4 after scratching. C. Migration distance of A2780 cells in the REV7 overexpression group and control group at day 2 and day 4 after scratching. All values ​​are expressed as mean ± SD. (*P<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0090] Figure 15 Effects of REV7 gene silencing and overexpression on apoptosis in A2780 and SKOV3 cells. AB. Silencing the REV7 gene with REV7 siRNA1 and pcDNA3.1 REV7 overexpression: After 48 hours of REV7 gene overexpression, apoptosis was detected by flow cytometry after Annexin V-FITC / propidium iodide (PI) staining. The distribution of normal viable cells, early apoptotic cells, late apoptotic cells, and dead cells was shown by scatter plots of PI (y-axis) and Annexin V (x-axis). CD. The percentages of normal viable cells, early apoptotic cells, late apoptotic cells, and dead cells were calculated. Data are presented as the mean ± SD of three experiments (*P<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0091] Figure 16 : Schematic diagram of the epigenetic editing design of the REV7 promoter.

[0092] Figure 17 Epigenetic editing validation of the REV7 promoter. AC. Changes in DNA methylation around the sgRNA binding site in OC cells A2780 (A), SKOV3 cells (B), and 293T cells (C) 48 hours after transfection. The horizontal axis represents the chromosomal location of the CG site, and the vertical axis represents the methylation level at that site. D. Expression level of the REV7 gene in OC cells after promoter epigenetic editing, expressed as mean ± SD. Statistical analysis was performed using a two-tailed Student's t-test (*p<0.05, **p<0.01).

[0093] Figure 18The effect of REV7 promoter epigenetic editing on the HR repair capacity of A2780 cells. A. Dual immunofluorescence staining of RAD51 / γH2AX in A2780 cells exposed to 0.75 μg / ml DDP or 10 μM Olaparib. Scale bar is 5 μm. B. Quantification of γH2AX and RAD51 by immunofluorescence focusing in up to 100 cells per group of A2780 cells. γH2AX protein is green fluorescent, indicating intracellular DNA damage, and RAD51 is red fluorescent, indicating intracellular DNA repair.

[0094] Figure 19 The effect of REV7 promoter epigenetic editing on the HR repair capacity of SKOV3 cells. A. Dual immunofluorescence staining of RAD51 / γH2AX in SKOV3 cells exposed to 0.75 μg / ml DDP or 100 μM Olaparib. Scale bar: 5 μm. B. Quantification of γH2AX and RAD51 by immunofluorescence focusing in up to 100 cells per group of SKOV3 cells. γH2AX protein showed green fluorescence, indicating intracellular DNA damage, while RAD51 showed red fluorescence, indicating intracellular DNA repair.

[0095] Figure 20 The effect of epigenetic editing of the REV7 promoter on the sensitivity of OC cells to HR drugs. AB. Proliferation curves of A2780 cells treated with 0.75 μg / ml cisplatin or 10 μM Olaparib for 4 days after epigenetic editing, expressed as OD450. CD. Bar plots showing the OD450 of A2780 and SKOV3 cells after 4 days of drug treatment, expressed as mean ± SD. Detailed Implementation

[0096] Through in-depth research, the inventors designed an editing method targeting a specific REV7 promoter region. Validation of the editing effects revealed that epigenetic inactivation of REV7 in OC cells significantly increased sensitivity to platinum-based and PARP inhibitors.

[0097] This invention provides a kit or composition comprising (1) sgRNA or its complementary sequence, or a nucleic acid construct containing said sgRNA or its complementary sequence, and (2) an epigenetic editing reagent. This kit or composition can increase the methylation level of the TSS region of the REV7 promoter at positions 11751554 to 11751655, or positions 11752480 to 11752544.

[0098] In this paper, the "TSS region" refers to the transcription start site of a gene. This region typically contains gene promoters, regulatory elements, and transcription factor binding sites. In this application, REV7 is located at 1p36.22, and the TSS is located at Chr1.11751707. Epigenetic editing is performed on a DNA region or fragment thereof from 2,000 bp upstream to 1,000 bp downstream of the REV7 promoter region 11751707. The fragment of the REV7 promoter region is a segment from positions 11753707 to 11750708 on human chromosome 1, containing positions 11751554 to 11751655; or a segment from positions 11753707 to 11750708 on human chromosome 1, containing positions 11752480 to 11752544. In one or more embodiments, the REV7 promoter region, as shown in SEQ ID NO:20, corresponds to positions 11753707 to 11750708 of chromosome 1. The chromosome numbering in this document is based on the human reference genome GRCh37.

[0099] In one or more embodiments, increasing the methylation level of the REV7 promoter region or a fragment thereof refers to an increase in the average methylation level of any one or more or all CG dinucleotides in the sequence from positions 11751554 to 11751655 of human chromosome 1; or it refers to an increase in the average methylation level of any one or more or all CG dinucleotides in the sequence from positions 11752480 to 11752544 of human chromosome 1. Positions 11753707 to 11750708 of human chromosome 1 contain CG dinucleotides at the following positions: 11751669, 11751816, 11751916, 11752480, 11752483, 11752492, 11752494, 11752497, 11752538, 11752541, and 11752544.

[0100] In one or more embodiments, increasing the methylation level of the REV7 promoter region or a fragment thereof refers to increasing the methylation level at the following sites on human chromosome 1 from position 11753707 to 11750708: 11751669, 11751816, 11751916, 11752480, 11752483, 11752492, 11752494, 11752497, 11752538, 11752541, and 11752544.

[0101] In this article, "DNA methylation" or "methylation" can be used interchangeably, referring to the process of acquiring a methyl (CH3) group through covalent binding. This is a chemical modification process of S-adenosylmethionine (SAM), which acts as a CH3 donor, under the catalysis of DNMT at specific bases in the DNA sequence. Cytosine (C) is the most commonly methylated base in this process, and C-site methylation mainly occurs on CpG sequences. In mammals, some CpG dinucleotides are scattered throughout the genome, while others appear in dense clusters of CpG islands. CpG islands are usually unmethylated and are often located in promoter regions of genes that regulate transcription initiation and repression. Hypermethylation of CpG sites in enhancers or promoters usually leads to transcriptional silencing, while hypomethylation of CpG sites in the genome usually leads to gene expression activation. Therefore, methylation typically regulates gene expression through gene transcription. In this invention, the inventors discovered that methylating a specific region of the REV7 promoter using epigenetic editing tools can inactivate REV7, thereby improving drug sensitivity in ovarian cancer.

[0102] DNA methylation ratio is the proportion of methylated cytosine to cytosine, expressed as a percentage from 0% to 100%.

[0103] In this document, the calculation of "methylation level" can be performed in various ways known in the art. Exemplarily, the methylation level can be the proportion of cells with methylation at a given site to the total number of cells. The methylation level at multiple sites can be the mean, weighted mean, median, or obtained using methylation level calculation procedures known in the art.

[0104] In this article, epigenetic editing tools or epigenetic editing reagents may include the CRISPRoff system and sgRNA.

[0105] The CRISPRoff system includes DNA methyltransferases responsible for adding methyl groups to cytosine on DNA molecules, including DNMT1, DNMT2, DNMT3A, and DNMT3B. The CRISPRoff system may also include DNMT3L, which interacts with the DNMT3A protein to form a complex and enhances the methylation activity of DNMT3A on DNA. The DNA methyltransferases can fuse with nuclear localization signals to carry the fusion protein into the cell nucleus.

[0106] The CRISPRoff system also includes Cas enzymes, such as dCas9, which lacks endonuclease activity. Cas enzymes bind to sgRNA to recognize specific sites. The CRISPRoff system may also include the KRAB domain for further recruitment of DNA methyltransferases to increase DNA methylation levels and histone deacetylases to reduce H3 acetylation. Cas enzymes can fuse with nuclear localization signals to deliver the fusion protein into the nucleus.

[0107] The CRISPRoff system may also include peptide epitopes for antibody detection, such as those containing HA.

[0108] The CRISPRoff system may also include a fluorescent protein for observing the expression of the fusion protein; preferably, the fluorescent protein is BFP.

[0109] The CRISPRoff system can be a fusion protein comprising its components or its expression vector. For example, the fusion protein may comprise (1) a DNA methyltransferase, such as DNMT1, DNMT2, DNMT3A, or DNMT3B, or (2) a Cas enzyme, such as Cas9, preferably a Cas9 mutant dCas9 without endonuclease activity. The fusion protein may optionally further comprise a DNA-binding protein located at the N- or C-segment of the DNA methyltransferase, such as DNMT3L; optionally, a KRAB domain located at the N- or C-segment of the Cas enzyme; optionally, a nuclear localization signal; and optionally, a marker for detection, such as a peptide epitope or a fluorescent protein. The domains or components of the fusion protein may or may not have linkers, such as XTEN80.

[0110] In one or more embodiments, the CRISPRoff system comprises a fusion protein or its expression vector, the fusion protein comprising, from the N-terminus to the C-terminus, DNMT3A, DNMT3L, XTEN80, dCas9, HA, NLS, BFP, and KRAB. An exemplary CRISPRoff plasmid was purchased from Addgene.

[0111] sgRNA is a guide RNA and an important component of the CRISPR gene knockout / knockin system. It consists of two parts: tracRNA and crRNA. When these two parts are fused and expressed, the sgRNA can effectively perform its guide function, binding to the Cas9 protein and guiding the Cas9 enzyme to target and cleave genomic DNA. In some embodiments, the sgRNA described herein has the sequence shown in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

[0112] This invention also provides the use of substances (including sgRNA and epigenetic editing reagents) that increase the methylation level of the REV7 promoter region from positions 11751554 to 11751655 or from 11752480 to 11752544 in the preparation of a drug for treating ovarian cancer or for increasing the sensitivity of ovarian cancer cells to platinum-based drugs and / or PARP inhibitors. The ovarian cancer cells show upregulated REV7 expression or no significant change compared to the control, and the tumor cells show upregulated REV7 expression or no significant change compared to the control. The control can be a negative control commonly used in the art, such as REV7 expression in normal cells or tissues of the subject, REV7 expression in adjacent normal cells, or the average REV7 expression of a population. Ovarian cancer cells include SKOV3 cells or A2780 cells. In the drug, sgRNA, platinum-based drugs, and PARP inhibitors are all active ingredients.

[0113] "Platinum-based drugs" primarily refer to medications used to treat neoplastic diseases, including cisplatin, carboplatin, nedaplatin, oxaliplatin, and lobaplatin. In some embodiments, the platinum-based drug is cisplatin. "PARP inhibitors," also known as poly(ADP-ribose) polymerase inhibitors, can affect the self-replication of cancer cells. These include olaparib, niraparib, fluzoparib, and pamiparib. In some embodiments, the PARP inhibitor is olaparib.

[0114] In addition to these active ingredients, the drug also contains pharmaceutically acceptable excipients. The term "pharmaceutically acceptable excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, and is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995). Pharmaceutically acceptable excipients include, but are not limited to, diluents, carriers, solubilizers, emulsifiers, preservatives, and / or adjuvants. The excipients are preferably non-toxic to the recipient at the dosage and concentration used. Such excipients include, but are not limited to, saline, buffers, glucose, water, glycerol, ethanol, and combinations thereof. In some embodiments, the drug may contain substances for improving, maintaining, or retaining, for example, the pH, permeability, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption, or permeation of the composition. These substances are known in the art. The optimal drug can be determined based on the expected route of administration, delivery method, and required dosage.

[0115] Medications intended for internal administration are typically provided in sterile formulations. Sterilization is achieved by filtration through a sterile filter membrane. When the composition is lyophilized, sterilization can be performed using this method before or after lyophilization and rehydration. Medications of the present invention may be used for parenteral delivery. Medications for parenteral administration may be in lyophilized form or stored in solution. They are prepared, for example, using physiological saline or aqueous solutions containing glucose and other excipients by conventional methods. Parenteral medications are typically placed in containers with sterile access openings, such as intravenous solution bands or vials with stoppers that can be punctured by a hypodermic needle. Alternatively, medications may be intended for inhalation or delivery via the digestive tract (e.g., orally). The preparation of the pharmaceutically acceptable medications is within the scope of the art. Other medications will be apparent to those skilled in the art. Techniques for formulating a variety of other continuous or controlled delivery methods (such as liposome carriers, bioeasily perishable microparticles or porous beads and accumulation injection) are also known to those skilled in the art.

[0116] Once formulated, the drug is stored in sterile vials in the form of a solution, suspension, gel, emulsion, solid, crystal, or dehydrated or lyophilized powder. The formulation may be stored in a ready-to-use form or in a rehydrated form prior to application (e.g., lyophilized).

[0117] The present invention also provides a method for treating cancer, comprising administering a therapeutically effective amount of an agent that inhibits REV7 gene expression and a therapeutically effective amount of an anticancer drug. Exemplarily, the agent that inhibits REV7 gene expression is an agent described herein that increases the methylation level of the REV7 promoter region or a fragment thereof, such as sgRNA and optionally the CRISPRoff system.

[0118] In this application, the reagent for inhibiting REV7 gene expression is a repressive molecule that specifically interferes with the transcription and / or expression of the REV7 gene. In one or more embodiments, the repressive molecule targets the REV7 gene or its transcripts. In one or more embodiments, the repressive molecule is selected from the group consisting of: (a) small molecule compounds, antisense nucleic acids, microRNA, siRNA, RNAi, dsRNA, sgRNA, antibodies, or combinations thereof, and (b) nucleic acid constructs capable of expressing or forming (a). Exemplarily, the repressive molecule of this application has any of the sequences shown in SEQ ID NO:8-13.

[0119] In one or more embodiments, the inhibitory molecule has the sequences shown in SEQ ID NO:8 and 9. In one or more embodiments, the inhibitory molecule has the sequences shown in SEQ ID NO:10 and 11. In one or more embodiments, the inhibitory molecule has the sequences shown in SEQ ID NO:12 and 13. Preferably, the inhibitory molecule has the sequences shown in SEQ ID NO:8 and 9. In one or more embodiments, the inhibitor further comprises a Cas enzyme (e.g., Cas9), its coding sequence, and / or a nucleic acid construct expressing the Cas enzyme.

[0120] The reagent for inhibiting REV7 gene expression and the anticancer drug can be administered sequentially, at intervals, or simultaneously. At intervals refer to administering the anticancer drug at intervals between administration of the reagent for inhibiting REV7 gene expression or at intervals between administration of the anticancer drug. In one or more embodiments, the anticancer drug is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after administration of the reagent for inhibiting REV7 gene expression.

[0121] "Treatment" refers to the use of the treatment regimens described herein by a subject to achieve at least one positive therapeutic effect (e.g., a reduction in the number of cancer cells, a decrease in tumor volume, a reduction in the rate of cancer cell invasion into surrounding organs, or a reduction in the rate of tumor metastasis or growth). Effective treatment regimens for patients can vary depending on various factors, such as the patient's disease state, age, weight, and the ability of the therapy to elicit an anti-cancer response in the subject. The therapeutically effective amount of the reagents containing the present invention that increase the methylation level of the REV7 promoter region will depend, for example, on the extent and target of treatment. Those skilled in the art will understand that the appropriate dose level for treatment will vary in part depending on the delivered molecule, indication, route of administration, and the patient's size (weight, body surface or organ size) and / or condition (age and general health status). In some embodiments, clinicians may titrate the dose and change the route of administration to obtain optimal therapeutic effects. For example, approximately 10 micrograms / kg body weight to approximately 50 milligrams / kg body weight per day.

[0122] The frequency of administration will depend on the pharmacokinetic parameters of the substance in the formulation used. Clinicians typically administer drugs containing the substance until a dose is reached to achieve the desired effect. The drug can therefore be administered as a single dose, or over time as two or more doses (containing or not containing the same amount of the desired molecule), or via implanted device or catheter as a continuous infusion.

[0123] The route of administration of the drug is based on known methods, such as oral, intravenous, intraperitoneal, intracerebral (within the brain parenchyma), intraventricular, intramuscular, intraocular, intraarterial, portal vein or intralesional injection; via a continuous release system or via an implanted device.

[0124] The present invention also provides a non-therapeutic method for reducing REV7 expression in cells in vitro, including agents that increase the methylation level of the REV7 promoter region or fragments thereof, or agents that inhibit REV7 gene expression.

[0125] This invention also provides a method and reagents for increasing the sensitivity of ovarian cancer cells to platinum-based drugs and PARP inhibitors. The method primarily involves epigenetic editing of the REV7 promoter to alter the response of ovarian cancer cells to platinum-based drugs. This editing can be achieved using the specific epigenetic editing tool CRISPRoff.

[0126] Specifically, the method of the present invention includes the following steps: 1. Designing and synthesizing the epigenetic editing tool CRISPRoff. 2. Designing and synthesizing a guide RNA to direct the tool to perform specific editing of the REV7 promoter. 3. Introducing the tool and guide RNA into ovarian cancer cells to edit the REV7 promoter. 4. Verifying the editing effect and whether the sensitivity of the edited ovarian cancer cells to platinum-based drugs is increased through experimental methods.

[0127] Furthermore, this invention also provides a reagent for carrying out the above-described method. This reagent includes a guide RNA (gRNA) for directing the tool to edit the REV7 promoter. Using the method and reagent of this invention, the sensitivity of ovarian cancer cells to platinum-based drugs and PARP inhibitors can be effectively increased, thereby improving the efficacy of drug treatment for ovarian cancer.

[0128] Example

[0129] Materials and methods

[0130] 1. Clinical Sample Collection

[0131] We collected 134 formalin-fixed paraffin-embedded (FFPE) ovarian cancer (OC) samples and 17 normal ovarian control samples from Xiangya Hospital, School of Basic Medical Sciences, Central South University (Changsha, China). Survival information was available for 67 OC samples, and matched peripheral blood samples from 42 high-grade serous ovarian cancer (HGSOC) samples were available for HRD analysis. Our study received ethical approval from the local committee, and all participants provided written informed consent.

[0132] 2. HRD Score Calculation

[0133] Based on the previous description [Marquard AM, Eklund AC, Joshi T, et al. Biomark Res. 2015; 3:9. doi:10.1186 / s40364-015-0033-4], genomic scar features were analyzed using the R program to calculate the HRD score. The calculation included determining scores for telomere allelic imbalance (TAI), large-scale variation (LST), and loss of heterozygosity (LOH). TAI indicates an allelic imbalance region extending into the telomere, LOH indicates a chromosomal LOH region longer than 15 Mb, and LST refers to the breakpoint between regions longer than 10 Mb after filtering out regions shorter than 3 Mb. The HRD score was calculated by summing the TAI, LST, and LOH scores.

[0134] 3. Bisulfite amplicon sequencing (BSAS)

[0135] Genomic DNA was extracted from normal ovarian tissue samples and ovarian cancer cell samples using the High Pure PCR Template Preparation Kit (Roch, 11796828001) and following the instructions. GeneRead was used to extract the DNA. TM Genomic DNA was extracted from FFPE samples of ovarian cancer tissue using the DNA FFPE Kit (QIAGEN, 180134) and following the manufacturer's instructions. DNA methylation levels of candidate genes were assessed using bisulfite amplification and next-generation sequencing (NGS). Specifically, 10 ng of genomic DNA was bisulfite-converted using the EZ DNA Methylation-Lightning Kit (Zymo Research, USA). Subsequently, the target sequences were amplified by PCR, and a second-generation sequencing library was constructed using the TaKaRaEX Taq Hotstart Kit (China). Methylation-independent PCR / MIP primers were designed using MethPrimer 2.0 software. The primer sequences are as follows: REV7 clinical sample validation primer (forward (SEQ ID NO:4): 5'-GAGTGYGTTYGGAATTAGTAT-3', reverse (SEQ ID NO:5): 5'-AAACCACAAARGAAAAACCAA-3'); REV7 methylation editing validation primer (forward (SEQ ID NO:6): 5'-GGGTTTGATTGTTTTTAGTT-3', reverse (SEQ ID NO:7): 5'-TTATCTACCTCCTTTATCTACCCCA-3').

[0136] The quality of sequencing data was validated using the FastQC software package on the NGS HiSeq platform (Illumina, Inc.). Aptamer removal and quality trimming were performed using Cutadapt v1.9.1, and reads were aligned to a reference genome (UCSC hg19) using Bismark. The methylation level of individual cytosines was determined using the R software package MethylKit v1.26.0.

[0137] 4. Survival analysis

[0138] The correlation between REV7 methylation levels and survival was estimated using the Kaplan-Meier (KM) method with the Survminer (version 0.4.9, https: / / cran.r-project.org / ) R software package. The log-rank test was used for survival comparisons. A p-value < 0.05 was considered significant.

[0139] 5. Cell Culture and Drug Treatment

[0140] Six OC cell lines were purchased: A2780, A2780-DDP, CoC1, CoC1-DDP, OVCAR3, SKOV3, and 293T. Among these, A2780 and A2780-DDP, and CoC1 and CoC1-DDP were two pairs of drug-resistant strains. 293T cells were cultured in Dulbecco's Modified Eagle Medium (DMEM), while the OC cell lines A2780, CoC1, OVCAR3, and SKOV3 were cultured in RPMI-1640 medium. A2780-DDP and CoC1-DDP cultures were supplemented with 2 μg / ml cisplatin (DDP) to maintain cell resistance. These cell cultures were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (P / S) antibiotics. Culture conditions included maintaining cells at 37°C and ensuring appropriate humidity in an environment containing 5% CO2. 1 μM azacytidine (Sigma) treatment: A2780 and SKOV3 cells were plated the day before, and when the cells reached 70%-80% confluence 24 hours later, they were treated with 1 μM azacytidine for 48 hours before the cells were collected for subsequent experiments. REV7 siRNA drug treatment, the siRNA sequences are as follows: REV7 siRNA1 (forward (SEQ ID NO:8): 5'-AAGAUGCAGCUUUACGUGGAAdTdT-3', reverse (SEQ ID NO:9): 5'-UUCCACGUAAAGCUGCAUCUUdTdT-3'); REV7 siRNA2 (forward (SEQ ID NO:10): 5'-UGCUGUGAGUUGUUUCAAUAAAGdGdG-3', reverse (SEQ ID NO:11): 5'-CCCUUUAUUGAAACAACUCACAGCACA-3'); REV7 siRNA3 (forward (SEQ ID NO:12): 5'-GAUGCAGCUUUACGUGGAAdTdT-3', reverse (SEQ ID NO:13): 5'-UUCCACGUAAAGCUGCAUCdTdT-3').

[0141] 6. Plasmid construction

[0142] The eukaryotic expression vectors pcDNA3.1 and pEGFP-C3 plasmid were obtained from laboratory storage, with pEGFP-C3 containing the enhanced green fluorescent protein (EGFP) reporter gene. Total RNA was extracted from A2780 cells using reverse transcription-polymerase chain reaction (RT-PCR), and the full-length cDNA of the REV7 gene was amplified. This gene was then ligated into pcDNA3.1 and pEGFP-C3 via Xho I and BamHI restriction sites.

[0143] The plasmids hU6-sgRNA-hUbC-dCas9-KRAB-T2a-GFP and hU6-sgRNA-hUbC-dCas9-KRAB-T2a-Puro were purchased from Addgene (Addgene #71236 and #71237). Both plasmids contain the dCas9-KRAB functional region and can be used with the BsmBI restriction site to generate the target sgRNA via restriction cloning of the protospacer downstream of the U6 promoter. The former contains a green fluorescent protein (GFP) marker for measuring transfection efficiency, while the latter contains a puromycin selection gene for positive selection. The sgRNA sequences targeting the REV7 promoter are as follows: (SEQ ID NO:1) 5'-GCCCGCGCACCTCTCCACGC-3', targeting position 11751816 on chromosome 1; (SEQ ID NO:2) 5'-CCGGCCGGACAGGTAAGGCA-3', targeting position 11751916 on chromosome 1; (SEQ ID NO:3) 5'-GCTCGACTGCCCCCAGCCGA-3', targeting position 11751669 on chromosome 1.

[0144] 7. Virus Packaging and Infection

[0145] When 293T cells reached 70%-80% confluence, 9 μg pLV hU6-sgRNA hUbC-dCas9-KRAB-T2a-Puro plasmid, 6 μg VSVG plasmid, and 5 μg PSPAX2 plasmid were added to a 10 cm cell culture dish. Virus packaging was then performed via Lipofectamine 3000 transfection. Three days later, the culture medium was collected, and the virus was concentrated using a 100K concentration tube (MERCK) at 4500×g for 30 min, and then aliquoted and stored at -80℃. When cells reached 30%-40% confluence after 24 h of culture, 293T, A2780, and SKOV3 infections were performed using an MOI of 5.

[0146] 8. Identification of transcriptional activity

[0147] The pCpGL-basic vector was obtained from Shanghai Medical College of Fudan University, and the pRL vector was preserved in the laboratory. Primers with two REV7 promoter regions containing HindIII and BamHI restriction sites were designed using Primer5 software. The primer sequences are as follows: forward (SEQ ID NO:14): 5'-CGCGGATCCGGACTGGCGGATAGACGGGT-3', reverse (SEQ ID NO:15): 5'-CCCAAGCTTGCCTCGCTCCATTGTTGGGG-3'). The vector and primers were digested with the above enzymes and then ligated with T4 ligase. The plasmid was methylated in vitro using CpG methyltransferase M.Sss I (NEB), and the methylation level of the constructed plasmid was detected using the methylation-sensitive restriction endonuclease HpaII. 293T cells were seeded in 96-well plates. After 24 hours, methylated / unmethylated pCpGL-REV7-promoter and pRL vector were co-transfected with Lipofectamine 3000. The effect of methylation on the transcriptional activity of the target gene promoter was detected using a dual-luciferase reporter gene assay kit (Yisheng). The specific operation was performed according to the instructions.

[0148] 9. Real-time quantitative polymerase chain reaction

[0149] Total RNA was extracted from cultured cells and tissue samples using TRIzol reagent (Sigma, USA). Reverse transcription of mRNA was performed using the PrimeScript RT kit (TaKaRa, China) with a mixture of oligo-dT and random primers. qPCR was performed using the standard three-step amplification protocol of SYBR Premix Ex Taq™ (TaKaRa, China). qPCR reactions were performed using a CFX96 Real-Time PCR instrument (Bio-Rad, USA) and SYBR Green PCR Mixture (TaKaRa, China). Changes were calculated using the comparative Cq method. Amplification conditions were 60°C for 60 min, followed by 95°C for 5 min, 95°C for 15 sec, and 60°C for 1 min, for 45 PCR cycles. REV7 primers (forward (SEQ ID NO:16): 5'-CCAGGCTGTACCTTCACAGTC-3, reverse (SEQ ID NO:17): 5'-TCTTCCACGTAAAGCTGCATC-3'), GAPDH primers (forward (SEQ ID NO:18): 5'-TCATTGACCTCAACTACATGGTTT-3', reverse (SEQ ID NO:19): 5'-GAAGATGGTGATGGGATTTC-3').

[0150] 10. Western blot detection

[0151] Cells were lysed using RIPA lysis buffer (Yisheng) and protein concentration was determined using a BCA protein assay kit (Thermo Fisher). Proteins were subjected to SDS-PAGE gel electrophoresis and transferred to a PVDF membrane. Immunohistometry was then performed using REV7 antibody (CST) and control GAPDH antibody, and imaging was performed using a G:BOX Chemi XX9 all-purpose biological imaging system (G:BOX).

[0152] 11.CCK8

[0153] Twenty-four hours before transfection, A2780 and SKOV3 cells were seeded into 6-well plates to achieve 70%-90% confluency. 100 nM REV7 siRNA / NC siRNA was transfected using Lipofectamine 3000 with an MOI of 5. Non-editing virus was used as a control. Cells were harvested 24 hours after transfection / infection. A2780 and SKOV3 cells were seeded at 2 × 10⁶ cells per well. 4 The sum of 4 × 10 3 Cells were seeded at a density of [insert seeding density here] into 96-well plates. After 24 hours of culture, cisplatin (DDP) (0.75 μg / ml) (Sigma, USA) and olaparib (A2780 10 μM and SKOV3 100 μM) (MedChemExpress, USA) were added for drug sensitivity testing. 10 μl of cell counting reagent (DOJINDO, China) was added on days 0, 1, 2, 3, and 4 after drug administration. After 2 hours of culture, the optical density (OD450) at 450 nm was measured to analyze cell proliferation.

[0154] 12. Cloning experiments

[0155] After 24 hours of plating with A2780, SKOV3, and OVCAR3 plasmids, cells were transfected with pcDNA3.1-REV7, pEGFP-REV7, and a control plasmid, respectively. 24 hours after transfection, cells were trypsinized, counted to 100 cells, and seeded into 6-well plates. Cells were cultured for 14 days, with the medium changed every 2 days. After colony formation, cells were fixed with 4% paraformaldehyde for 15-30 minutes, washed with PBS, stained with crystal violet, and photographed for cell counting.

[0156] 13. Scratch test

[0157] After A2780 cells were seeded in 6-well plates for 24 hours, they were transfected with 100 nM REV7 siRNA1 and control siRNA, and 500 ng pcDNA3.1-REV7 and control plasmids, respectively. Cells were scratched 24 hours after transfection, and images were taken on the day of scratching, day 2, and day 4. Cell migration distance was measured using ImageJ software.

[0158] 14. Homologous Recombination (HR) Detection

[0159] To assess the HR capacity of A2780 and SKOV3 cells, dual immunofluorescence staining of γH2AX and RAD51 was performed. The REV7 gene was generated by infecting cells with hU6-sgREV7-hUbC-dCas9-KRAB-T2a-Puro virus and a control virus. After 2 days of culture, cells were exposed to DDP (1 μg / ml) and Olaparib (10 μM for A2780 and 100 μM for SKOV3) for 24 hours. Subsequently, nuclei were extracted on ice for 10 minutes with a buffer containing 20 mM HEPES (pH 7.4), 20 mM NaCl, 5 mM MgCl2, 0.5% NP-40, 1 mM DTT, and a protease inhibitor. Cells were then washed with ice-cold PBS and fixed with 4% PFA at room temperature for 10 minutes. Cells were then blocked for 30 minutes in an immunofluorescence (IF) blocking buffer consisting of 10% goat serum, 0.5% NP-40, and 0.5% saponin in PBS. They were then incubated for 2 hours at room temperature with primary antibodies diluted in the blocking buffer: mouse anti-RAD51 (GeneTex, USA) and rabbit anti-γ-H2AX (Cell Signaling, USA). After primary antibody incubation, cells were washed with PBS (3 × 5 min) and stained with fluorescent secondary antibodies: goat anti-rabbit IgG (H+L) secondary antibody, DyLight488 (Thermo Fisher, USA), and goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 555 (Thermo Fisher, USA) at room temperature for 1 hour. After secondary antibody staining, cells were washed as described above, mounted on ProLong Gold slide media (Thermo Fisher, USA), and imaged using Ti-E+A1R+STORM10-1 (Nikon, Japan). In two independent experiments, data from 100 cells were collected in 5 to 10 fields of view.

[0160] 15. Apoptosis detection

[0161] After trypsin digestion of adherent cells with EDTA-free trypsin, cells were collected by centrifugation at 300×g for 5 minutes at 4°C. Apoptosis rate was determined using a cell cycle and apoptosis assay kit (Yisheng, China). Specifically, cells were washed twice with pre-cooled PBS. Approximately 1-5 × 10⁶ cells were collected. 5Cells were collected. PBS was removed, and cells were resuspended in 100 μl of 1× binding buffer. 5 μl of Annexin V-FITC and 10 μl of PI staining solution were added and gently mixed. The mixture was incubated in the dark at room temperature for 10–15 minutes. 400 μl of 1× binding buffer was added and thoroughly mixed. The sample was then placed on ice and analyzed by flow cytometry within 1 hour.

[0162] Experimental results

[0163] 1. Significant changes in REV7 promoter methylation were observed in clinical samples of ovarian cancer.

[0164] We detected the methylation level of the REV7 promoter region DNA in normal ovarian tissue samples, ovarian cancer tissue samples, and ovarian cancer cell samples from healthy ovarian individuals. We used BSAS to validate the differentially methylated region of the REV7 promoter obtained from previous RRBS sequencing analysis. The REV7 gene TSS site is located at Chr1.11751707, and the validation region is located 800 bp upstream of the TSS site, with a fragment length of 108 bp. This region is part of a CpG island containing 8 CpG sites. The results showed significant differences in methylation at the validation site in the REV7 promoter region. Figure 1 A comparison of the average methylation levels at eight sites in the target fragment revealed that the REV7 promoter methylation level in ovarian cancer cells and tissues (70.85% and 34.09%, respectively) was significantly higher than that in normal ovarian tissue (9.81%). These results validate the significant differences between ovarian cancer cells / tissues and normal ovarian tissues, suggesting its potential for clinical application in ovarian cancer screening. Figure 1 ).

[0165] We also detected the DNA methylation level of the REV7 gene target region in 74 FFPE samples from the primary ovarian cancer group and 13 FFPE samples from the recurrent ovarian cancer group. The results showed that the average methylation level of the REV7 promoter target region in recurrent ovarian cancer tissue (25.22%) was significantly lower than that in primary ovarian cancer tissue (42.63%). Figure 2 A), and the methylation levels of recurrent ovarian cancer tissue at multiple sites in this region are significantly lower than those of primary ovarian cancer tissue. Figure 2 The differences were statistically significant (p<0.05). These results indicate that the methylation status of the REV7 gene target fragment has the potential to serve as an indicator for ovarian cancer recurrence.

[0166] In addition, we collected 67 samples of ovarian cancer patients with long-term (up to 140 months) survival data and examined the impact of the mean methylation level of the REV7 promoter target region on the survival of ovarian cancer patients. The results showed that high methylation was associated with higher survival (p<0.05). Figure 3 ).

[0167] 2. REV7 promoter methylation is associated with homologous recombination defects.

[0168] HRD (human ovarian cancer) leads to defects in the repair pathway of DNA double-strand breaks, resulting in high sensitivity to platinum-based chemotherapy drugs and PARP inhibitors. Therefore, HRD has become a key biomarker for ovarian cancer treatment. This study analyzed the correlation between REV7 promoter methylation and HRD score using 42 clinical ovarian cancer samples with HRD scores, predicting the methylation status of the target fragment of the REV7 gene as an indicator of clinical ovarian cancer HRD score and platinum sensitivity. Comparison of REV7 promoter methylation levels between HRD-positive (HRD score ≥ 42) and HRD-negative (HRD score < 42) ovarian cancer samples showed significantly higher methylation levels in HRD-positive samples. Figure 4 ).

[0169] Furthermore, we observed a significant positive correlation between REV7 promoter methylation and HRD score. Figure 5 (p<0.05). These results further indicate that the REV7 promoter methylation level is associated with homologous recombination defects and may affect survival time through different responses to treatment, such as drug resistance, suggesting that the methylation status of the REV7 gene target fragment has the potential to serve as an indicator for clinical ovarian cancer HRD score and platinum sensitivity / resistance.

[0170] 3. REV7 gene promoter methylation regulates gene expression.

[0171] After 48 hours of treatment with 1 μM azacytidine in OC cells A2780, SKOV3, and OVCAR3, the expression level of REV7 in all three ovarian cancer cell lines significantly increased. Figure 6 The results (A) indicate that REV7 expression is regulated by methylation. Using a dual-luciferase reporter gene assay, the effect of REV7 promoter methylation on transcriptional activity was detected. The results showed that the transcriptional activity of the methylated promoter was only 32% of that of the unmethylated promoter. Figure 6 (B) indicates that the expression of the REV7 gene is directly regulated by its promoter methylation.

[0172] 4. Knockdown and overexpression of the REV7 gene

[0173] Three siRNAs were used to silence the REV7 gene in A2780, SKOV3, and 293T cells. qPCR results showed that the three siRNAs all produced varying degrees of knockdown effect on REV7 gene expression in different cell types. Figure 7Among them, REV7 siRNA1 showed the best knockdown effect in multiple cell types, achieving knockdown rates of 78.5%, 96.2%, and 86.0% in A2780, SKOV3, and 293T cells, respectively. Western blotting results showed that after siRNA1 knocked down the REV7 gene, the protein expression level of REV7 in A2780 and SKOV3 cells decreased by more than 80%. Figure 8 The silencing effect was significant, so this siRNA was selected for subsequent experiments.

[0174] Two expression plasmids, pcDNA3.1-REV7 and pEGFP-REV7, were constructed. Western blot results showed that the pcDNA3.1-REV7 expression plasmid increased the expression level of REV7 protein in A2780 and SKOV3 by more than 10-fold. These two systems can be effectively used for subsequent in vitro experimental studies.

[0175] 5. Immunofluorescence detection of the effects of REV7 siRNA and pcDNA3.1-REV7 on A2780 DNA damage and repair

[0176] After confirming that REV7 siRNA and pcDNA3.1-REV7 significantly altered REV7 gene expression, we investigated changes in cellular regenerative heart rate (HR) repair capacity and its sensitivity to platinum-based drugs and PARPi inhibitors. A2780 and SKOV3 cells were transfected with REV7 siRNA and pcDNA3.1-REV7 for 24 hours, then treated with 0.75 μg / ml DDP and 10 μM / 100 μM Laparib, respectively. HR capacity was measured by γH2AX / RAD51 double immunofluorescence staining one day later. γH2AX lesions represented locations of double-strand breaks on chromosomes, and RAD51 lesions represented ongoing HR repair. The ratio of the two co-localizations represented HR repair capacity. Results showed that OC cells A2780 (… Figure 9 ) and SKOV3 ( Figure 10 In this study, DDP caused more severe cell damage than Olaparib, while REV7 siRNA significantly reduced RAD51 lesions in cells. Overexpression of REV7 did not significantly change or slightly increased the number of RAD51 lesions in cells. Figure 11 ).

[0177] The counting results showed that the average number of RAD51 lesions in DDP-treated A2780 cells knocked down by REV7 siRNA was 115.8, while the average number of RAD51 lesions in the control group was 146.0. Figure 9(B); The average number of RAD51 lesions in cells treated with Olaparib was 83.8, while the average number of RAD51 lesions in the control group was 129.4. Figure 9 C).

[0178] In SKOV3 cells, the average number of RAD51 lesions in DDP-treated REV7 siRNA knockdown cells was 83.2, while the average number of RAD51 lesions in the control group was 218.9. Figure 10 (B); The average number of RAD51 lesions in cells treated with Olaparib was 67.7, while the average number of RAD51 lesions in the control group was 165.6. Figure 10 These results indicate that REV7 siRNA knockdown reduces HR repair capacity.

[0179] We also assessed the HR repair capacity of REV7-overexpressing SKOV3 cells. The average number of RAD51 lesions in DDP-treated REV7-overexpressing cells was 184.5, while the average number of RAD51 lesions in the control group was 178.9. Figure 11 (B); The average number of RAD51 lesions in cells treated with Olaparib was 169.2, while the average number of RAD51 lesions in the control group was 160.7. Figure 11 These results indicate that overexpression of REV7 did not significantly alter the repair capacity of HR.

[0180] 6. REV7 siRNA knockdown and overexpression affect cell proliferation.

[0181] To conduct drug sensitivity testing, A2780 and SKOV3 cells were transfected with REV7 siRNA for 48 hours. A2780 cells were then treated with 0.75 μg / ml DDP and 10 μM Olaparib for 4 days, while SKOV3 cells were treated with either 0.75 μg / ml DDP or 100 μM Olaparib for 4 days. Cell counting showed that REV7 siRNA treatment significantly reduced the proliferation of both A2780 and SKOV3 cells. Figure 12 Compared with the control group, the number of cells treated with REV7 siRNA decreased by 31.6% and 66.6% on day 4 after DDP treatment, respectively, while the number of cells treated with Olaparib decreased by 30.2% and 47.4%. Figure 12 (CD).

[0182] The above results indicate that REV7 siRNA knocks down the gene, inhibiting its expression and leading to a significant increase in cellular sensitivity to platinum-based drugs and PARPi drugs. In the absence of drug treatment, the number of A2780 and SKOV3 cells in the REV7 siRNA group decreased by 12.3% and 22.8% respectively on day 4, which was also significantly lower than that in the control group. Figure 12 (AB). This indicates that inhibiting REV7 gene expression can increase drug sensitivity by inhibiting OC cell proliferation.

[0183] 7. REV7 overexpression affects cell cloning ability.

[0184] After transfecting A2780, SKOV3, and OVCAR3 plasmids into pcDNA3.1, pcDNA3.1-REV7, and pEGFP-REV7 plasmids respectively, 100 cells were counted and reseeded into 6-well plates 24 hours later. The culture medium was changed every two days. After 14 days, the cells were fixed with 4% paraformaldehyde, stained with crystal violet, and photographed for cell counting (e.g., ...). Figure 13 ).

[0185] The results showed that in A2780 and OVCAR3 cells, cells transfected with pcDNA3.1-REV7 and pEGFP-REV7 plasmids, overexpressing REV7 protein, exhibited significantly enhanced cloning ability compared to cells transfected with the control plasmid pCDNA3.1. Transfection with both pcDNA3.1-REV7 and pEGFP-REV7 plasmids increased cloning ability by more than 100% compared to the control. In SKOV3 cells, cloning ability was poor after transfection with all three plasmids, with less than 10% of cells forming clones.

[0186] 8. REV7 siRNA knockdown and overexpression affect OC cell migration ability.

[0187] After A2780 cells were seeded in 6-well plates, they were transfected with NC siRNA, REV7 siRNA1, pcDNA3.1, and pcDNA3.1-REV7 for 24 hours. Scratching was performed 24 hours later, and images were taken on day 0 (scratching day), day 2 (scratching day), and day 4 (scratching day). The results showed that A2780 cells transfected with REV7 siRNA exhibited weaker migration ability compared to the control group (NC siRNA). On day 4, cells transfected with REV7 siRNA had an average migration distance of 536.9 μm, a decrease of 121.4 μm compared to the control group's average migration distance of 658.3 μm. Cells transfected with pcDNA3.1-REV7 showed enhanced migration ability compared to the control group (pcDNA3.1). On day 4, cells transfected with pcDNA3.1-REV7 had an average migration distance of 646.3 μm, an increase of 87.1 μm compared to the control group's average migration distance of 559.2 μm. The results are as follows: Figure 14As shown in the figure. It can be inferred that REV7 gene expression is closely related to the migration ability of OC cells, and reducing REV7 gene expression can inhibit the migration ability of OC cells.

[0188] 9. REV7 siRNA knockdown and overexpression affect OC cell apoptosis.

[0189] We analyzed the apoptosis of A2780 and SKOV3 cells 48 hours after REV7 siRNA knockdown and overexpression. Figure 15 Following REV7 siRNA knockdown, the apoptosis rates in A2780 and SKOV3 cells were significantly higher than those in control cells. Conversely, overexpression of the REV7 gene resulted in significantly lower apoptosis rates in A2780 and SKOV3 cells compared to control cells. These results suggest that decreased REV7 gene expression may promote apoptosis, while increased expression may inhibit it.

[0190] 10. Design and validation of the epigenetic editor for the REV7 promoter

[0191] The epigenetic editing plasmids hU6-sgRNA-hUbC-dCas9-KRAB-T2a-GFP and hU6-sgRNA-hUbC-dCas9-KRAB-T2a-Puro enhance DNA methylation and simultaneously produce the target sgRNA. The former exhibits green fluorescence, while the latter can be screened using puromycin. The inventors designed three sgRNAs (targeting chromosome 1 coordinates 11751816, 11751916, and 11751669, respectively), focusing particularly on the REV7 TSS region, and constructed the plasmids hU6-sgREV7-hUbC-dCas9-KRAB-T2a-GFP and hU6-sgREV7-hUbC-dCas9-KRAB-T2a-Puro for precise site-specific epigenetic modification. Figure 16 The viral vector was then packaged into a lentivirus to improve the epigenetic editing effect and achieve a long-lasting editing effect.

[0192] We infected 293T, A2780, and SKOV3 cells with hU6-sgREV7-hUbC-dCas9-KRAB-T2a-GFP virus. Subsequently, we observed the expression of the fluorescent protein on the plasmid using fluorescence microscopy for 7 days to assess expression efficiency and stability. The results clearly showed that fluorescent protein expression remained high on day 7, indicating that epigenetic editing has a long-term effect in cells. Stable transfected cells can then be obtained by puromycin selection using hU6-sgREV7-hUbC-dCas9-KRAB-T2a-pruo.

[0193] We further investigated the changes in DNA methylation levels after editing. Forty-eight hours after transfection, DNA methylation levels were significantly increased within a 400 bp range upstream and downstream of the sgRNA binding site in A2780, SKOV3, and 293T cells. Figure 17 DNA methylation editing was significantly effective, with methylation levels increasing by more than 80% in 293T cells, more than 60% in most CG cells in A2780 cells, and between 10% and 20% in most CG cells in SKOV3 cells.

[0194] Cells collected 48 hours after methylation editing were used to detect REV7 expression levels. qPCR results showed that methylation editing effectively silenced REV7 gene expression in different cell types. Figure 17 (D) The silencing effect was between 55% and 65%. These findings clearly demonstrate that epigenetic editing can effectively suppress REV7 expression in ovarian cancer.

[0195] 11. Immunofluorescence detection of the effects of epigenetic editing on DNA damage and repair in OC cells

[0196] After confirming that epigenetic editing significantly silenced REV7 gene expression, we investigated changes in cellular HR repair capacity and its sensitivity to platinum-based drugs and PARP inhibitors. A2780 cells were treated with 0.75 μg / ml DDP and 10 μM Olaparib for 24 hours after epigenetic editing, while SKOV3 cells were treated with either 0.75 μg / ml DDP or 100 μM Olaparib for 24 hours. HR capacity was then assessed using γH2AX / RAD51 dual immunofluorescence staining. γH2AX lesions represented locations of double-strand breaks on chromosomes, and RAD51 lesions represented ongoing HR repair. The ratio of the two co-localizations represented HR repair capacity. Results showed that OC cells A2780 treated with the drugs… Figure 18 ,A) and SKOV3( Figure 19 In A), DDP caused more severe cell damage than Olaparib, while RAD51 lesions were significantly reduced after REV7 epigenetic editing. Figure 18 ,BC Figure 19 ,BC).

[0197] The counting results showed that the average number of RAD51 lesions in A2780 cells treated with REV7 epigenetic editing was 127.1, while the average number of RAD51 lesions in the control group was 163.5. Figure 18(B); The average number of RAD51 lesions in cells treated with Olaparib was 84.2, while the average number of RAD51 lesions in the control group was 108.3. Figure 18 C).

[0198] In SKOV3 cells, the average number of RAD51 lesions in DDP-treated REV7 epigenetic editing cells was 96.1, while the average number of RAD51 lesions in the control group was 257.4. Figure 19 (B); The average number of RAD51 lesions in cells treated with Olaparib was 53.3, while the average number of RAD51 lesions in the control group was 196.9. Figure 19 These results indicate that REV7 epigenetic editing reduces HR repair capacity.

[0199] 12. REV7 epigenetic editing affects cell proliferation

[0200] To conduct drug sensitivity testing, after 48 hours of epigenetic editing of A2780 and SKOV3 cells using REV7, A2780 cells were treated with 0.75 μg / ml DDP and 10 μM Olaparib for 4 days, while SKOV3 cells were treated with either 0.75 μg / ml DDP or 100 μM Olaparib for 4 days. Cell counting showed that the proliferation of both A2780 and SKOV3 cells was significantly reduced after epigenetic editing treatment. Figure 20 Compared with the control group, the number of cells in the DDP treatment group decreased by 35.2% and 41.6% on day 4, respectively, while the number of cells in the Olaparib treatment group decreased by 20.7% and 38.1%, respectively. Figure 20 (CD).

[0201] The above results indicate that REV7 epigenetic editing suppressed gene expression, leading to a significant increase in cellular sensitivity to platinum-based drugs and PARPi. Compared with the unedited control group, the number of A2780 and SKOV3 cells in the epigenetic editing group decreased by 20.0% and 15.0% respectively on day 4, which was also significantly lower than that in the control group. Figure 20 (CD). This result suggests that REV7 epigenetic editing may increase drug sensitivity by inhibiting OC cell proliferation.

[0202] Part of the sequence in this article

[0203] SEQ ID NO:20-REV7 promoter region>NC_000001.10:c11753707-11750708Homosapiens chromosome 1,GRCh37.p13 Primary Assembly

[0204]

Claims

1. The use of reagents that inhibit REV7 gene expression in the preparation of drugs for treating cancer, drugs for improving the efficacy of anticancer drugs, and drugs for improving the sensitivity of tumor cells to anticancer drugs, wherein the anticancer drugs are chemotherapeutic drugs and / or PARP inhibitors, wherein, The reagent used to inhibit REV7 gene expression is: (1) A reagent that increases the methylation level of the REV7 promoter region or its fragments; or (2) Repressive molecules that specifically interfere with the transcription and / or expression of the REV7 gene. Preferably, The REV7 promoter region is the region from 2000 bp upstream to 1000 bp downstream of the transcription start site, and / or The chemotherapy drug in question is a platinum-based drug.

2. The use as described in claim 1, characterized in that, The REV7 promoter region is located at positions 11753707 to 11750708 on human chromosome 1. The segment of the REV7 promoter region is a segment of positions 11753707 to 11750708 on human chromosome 1, containing positions 11751554 to 11751655; or the segment of the REV7 promoter region is a segment of positions 11753707 to 11750708 on human chromosome 1, containing positions 11752480 to 11752544. Preferably, Increased methylation level in the REV7 promoter region or its segments refers to an increase in the average methylation level of one or more or all CG dinucleotides in the sequence from positions 11751554 to 11751655 on human chromosome 1, or Increased methylation level of the REV7 promoter region or its fragments refers to an increase in the average methylation level of one or more or all CG dinucleotides in the sequence from position 11752480 to position 11752544 of human chromosome 1.

3. The use as described in claim 1, characterized in that, The cancer in question is ovarian cancer, and the tumor cells are ovarian cancer cells.

4. The use as described in claim 1, characterized in that, The reagents used to increase the methylation level of the REV7 promoter region or fragments thereof comprise: (i) sgRNA or its complementary sequence, or (ii) nucleic acid constructs containing said sgRNA or its complementary sequence. Preferably, the sgRNA has a sequence as shown in SEQ ID NO:1, 2 and / or SEQ ID NO:3 or a variant having at least 80% sequence identity with it, and / or the reagent further comprises an epigenetic editing reagent. More preferably, the epigenetic editing reagent comprises a DNA methyltransferase, its coding sequence, or a nucleic acid construct expressing the Cas enzyme, and / or a Cas enzyme, its coding sequence, or a nucleic acid construct expressing the Cas enzyme.

5. The use as described in claim 4, characterized in that, The epigenetic editing reagent comprises a fusion protein or its expression vector, the fusion protein comprising: (1) DNA methyltransferases, such as DNMT1, DNMT2, DNMT3A, or DNMT3B, or (2) Cas enzymes, such as Cas9, Optionally, it may also contain DNA-binding proteins located in the N- or C-segment of DNA methyltransferases. Optionally, it may also include a KRAB domain located in the N or C segment of the Cas enzyme. The optional component also includes nuclear positioning signals. Optional features may also include biomarkers for detection, such as peptide epitopes and fluorescent proteins; Preferably, The fusion protein comprises, from N-terminus to C-terminus, the following: dCas9, KRAB structural domains dCas9, DNMT3A, DNMT3L, KRAB structural domain, dCas9, DNMT3A, DNMT3L, or dCas9, DNMT3A, DNMT3L, KRAB structural domains.

6. The use as described in claim 1, characterized in that, (2) The repressor molecule uses the REV7 gene or its transcript as the repressor target. Preferably, the inhibitory molecule is selected from the group consisting of: (a) small molecule compounds, antisense nucleic acids, microRNA, siRNA, RNAi, dsRNA, sgRNA, antibodies or combinations thereof, and (b) nucleic acid constructs that can express or form (a).

7. The use as described in claim 6, characterized in that, The inhibitory molecule has any of the sequences shown in SEQ ID NO:8-13. Preferably, the inhibitor further comprises a Cas enzyme (e.g., Cas9), its coding sequence, and / or a nucleic acid construct expressing the Cas enzyme.

8. Use of the reagent for inhibiting REV7 gene expression and the anticancer drug in the preparation of a medicament for treating cancer, wherein the reagent for inhibiting REV7 gene expression, the anticancer drug, and the anticancer drug are as described in any one of claims 1-7.

9. A non-therapeutic method for reducing REV7 expression in cells in vitro, comprising increasing the methylation level of the REV7 promoter region or a fragment thereof, wherein, The REV7 promoter region is located at positions 11753707 to 11750708 on human chromosome 1. The segment of the REV7 promoter region is a segment of human chromosome 1 from positions 11753707 to 11750708 containing positions 11751554 to 11751655, or... The REV7 promoter region is located at positions 11753707 to 11750708 on human chromosome 1, and the segment of the REV7 promoter region is a segment containing positions 11752480 to 11752544 within positions 11753707 to 11750708 on human chromosome 1. Preferably, increasing the methylation level of the REV7 promoter region or its fragment means increasing the average methylation level of any one or more or all CG dinucleotides in the sequence from position 11751554 to position 11751655 of human chromosome 1, or increasing the methylation level of the REV7 promoter region or its fragment means increasing the average methylation level of any one or more or all CG dinucleotides in the sequence from position 11752480 to position 11752544 of human chromosome 1.

10. A kit or composition comprising (1) an sgRNA or its complementary sequence targeting a fragment of the REV7 promoter region or thereof, or a nucleic acid construct containing said sgRNA or its complementary sequence, and (2) an epigenetic editing reagent, wherein, The REV7 promoter region is located at positions 11753707 to 11750708 on human chromosome 1. The segment of the REV7 promoter region is a segment of human chromosome 1 from positions 11753707 to 11750708 containing positions 11751554 to 11751655, or... The REV7 promoter region is located at positions 11753707 to 11750708 on human chromosome 1, and the segment of the REV7 promoter region is a segment containing positions 11752480 to 11752544 within positions 11753707 to 11750708 on human chromosome 1. Preferably, the nucleic acid construct is a cloning vector or expression vector of the sgRNA or its complementary sequence, and / or The sgRNA has a sequence as shown in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, or a variant having at least 80% sequence identity with it. The epigenetic editing reagent comprises a DNA methyltransferase, its coding sequence, or a nucleic acid construct expressing the Cas enzyme, and / or a Cas enzyme, its coding sequence, or a nucleic acid construct expressing the Cas enzyme.

11. The kit or composition according to claim 10, characterized in that, The epigenetic editing reagent comprises a fusion protein or its expression vector, the fusion protein comprising: (1) DNA methyltransferases, such as DNMT1, DNMT2, DNMT3A, or DNMT3B, or (2) Cas enzymes, such as Cas9, Optionally, it may also contain DNA-binding proteins located in the N- or C-segment of DNA methyltransferases. Optionally, it may also include a KRAB domain located in the N or C segment of the Cas enzyme. The optional component also includes nuclear positioning signals. Optional features also include markers for detection; Preferably, the fusion protein comprises, from the N-terminus to the C-terminus, the following: dCas9, KRAB structural domains dCas9, DNMT3A, DNMT3L, KRAB structural domain, dCas9, DNMT3A, DNMT3L, or dCas9, DNMT3A, DNMT3L, KRAB structural domains.

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