Reagent for improving methylation level of BRCA1 promoter and application

Abstract

The invention relates to a reagent for improving the methylation level of a BRCA1 promoter and application, and particularly provides an epigenetic editing reagent which enables the methylation level of a BRCA1 promoter region to be increased, so that BRCA1 is inactivated, and then ovarian cancer is treated and the drug sensitivity of the ovarian cancer is increased.

Description

TECHNICAL FIELD

[0001] The present application relates to epigenetics and tumor therapy, in particular to inactivation of BRCA1 by epigenetic editing to increase drug sensitivity of ovarian cancer. BACKGROUND

[0002] Drug resistance of ovarian cancer is a challenging problem that has a great impact on the prognosis of patients. Although the mechanisms behind this drug resistance have been extensively studied, effective strategies to reverse or sensitize drug-resistant tumors remain elusive. The limited progress in the treatment of ovarian cancer highlights the urgency of addressing the problem of drug resistance through innovative sensitization and drug methods.

[0003] Platinum-based chemotherapy is the gold standard for first-line treatment of ovarian cancer. Drug resistance that occurs during chemotherapy can be due to changes in genes and epigenetics, helping cancer cells to adapt to chemotherapy through various pathways such as stress, DNA damage, and apoptosis. Currently, epigenetic editing for chemotherapy sensitization faces some challenges, and it is still necessary to explore epigenetic regulatory sites for chemotherapy sensitivity. SUMMARY

[0004] To overcome the above-mentioned defects, the first aspect of the present application provides the use of an agent for inhibiting the expression of a BRCA1 gene in the preparation of a drug for treating cancer, a drug for improving the efficacy of an anticancer drug, and a drug for improving the sensitivity of tumor cells to an anticancer drug, wherein the anticancer drug is a chemotherapeutic drug and / or a PARP inhibitor.

[0005] In one or more embodiments, the agent for inhibiting the expression of the BRCA1 gene is an agent for increasing the methylation level of the BRCA1 promoter region or a fragment thereof.

[0006] In one or more embodiments, the BRCA1 promoter region is a region from 2000 bp upstream to 1000 bp downstream of the transcription start site.

[0007] In one or more embodiments, the BRCA1 promoter region is from position 41276500 to position 41279500 of human chromosome 17, and the fragment of the BRCA1 promoter region is a fragment containing position 41277060 to position 41277444 of human chromosome 17 from position 41276500 to position 41279500.

[0008] In one or more embodiments, the BRCA1 promoter region is as shown in SEQ ID NO: 11.

[0009] In one or more embodiments, increasing the methylation level of the BRCA1 promoter region or a fragment thereof refers to increasing the average methylation level of any one or more or all of the CG dinucleotides in the sequence from position 41277060 to position 41277444 of human chromosome 17.

[0010] In one or more embodiments, the cancer is a cancer in which BRCA1 expression is up-regulated, and the tumor cell is a tumor cell in which BRCA1 expression is up-regulated. In one or more embodiments, the cancer is not a cancer in which BRCA1 expression is down-regulated, and the tumor cell is not a tumor cell in which BRCA1 expression is down-regulated.

[0011] In one or more embodiments, the cancer is ovarian cancer, and the tumor cell is an ovarian cancer cell. In one or more embodiments, the cancer is ovarian cancer in which BRCA1 expression is up-regulated or not significantly changed, and the tumor cell is an ovarian cancer cell in which BRCA1 expression is up-regulated or not significantly changed.

[0012] In one or more embodiments, the tumor cell is a SKOV3 cell or an A2780 cell. In one or more embodiments, the tumor cell is a SKOV3 cell or an A2780 cell in which BRCA1 expression is up-regulated or not significantly changed.

[0013] In one or more embodiments, the chemotherapeutic drug is a platinum drug.

[0014] In one or more embodiments, the platinum drug comprises cisplatin, carboplatin, nedaplatin, oxaliplatin, lobaplatin, preferably, the platinum drug is cisplatin.

[0015] In one or more embodiments, the PARP inhibitor comprises olaparib, niraparib, talazoparib, pamiparib, preferably, the PARP inhibitor is olaparib.

[0016] In one or more embodiments, the agent for increasing the methylation level of the BRCA1 promoter region or a fragment thereof comprises: (1) an sgRNA or a complement thereof, or (2) a nucleic acid construct comprising the sgRNA or the complement thereof.

[0017] In one or more embodiments, the nucleic acid construct is a cloning vector or an expression vector of the sgRNA or the complement thereof.

[0018] In one or more embodiments, the sgRNA has a sequence as set forth in SEQ ID NO: 1 and / or SEQ ID NO: 2 or a variant thereof having at least 80% sequence identity thereto.

[0019] In one or more embodiments, the reagent further comprises an epigenetic editing reagent.

[0020] In one or more embodiments, the epigenetic editing reagent comprises a DNA methyltransferase or an expression vector thereof, and / or a Cas enzyme or an expression vector thereof.

[0021] 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, e.g., DNMT3L. Preferably, the N- or C- segment of the DNA methyltransferase is fused with DNMT3L.

[0022] In one or more embodiments, the Cas enzyme is Cas9, preferably a Cas9 mutant dCas9 without endonuclease activity. Preferably, the N- or C- segment of the Cas enzyme is fused with a KRAB domain.

[0023] In one or more embodiments, the epigenetic editing reagent further comprises a marker for detection or an expression vector thereof, e.g., a peptide epitope, a fluorescent protein.

[0024] In one or more embodiments, the epigenetic editing reagent comprises a fusion protein or an expression vector thereof, the fusion protein comprising

[0025] (1) a DNA methyltransferase, e.g., DNMT1, DNMT2, DNMT3A, or DNMT3B,

[0026] (2) a Cas enzyme, e.g., Cas9, preferably a Cas9 mutant dCas9 without endonuclease activity,

[0027] optionally further comprising a DNA binding protein, e.g., DNMT3L, located at the N- or C- segment of the DNA methyltransferase,

[0028] optionally further comprising a KRAB domain located at the N- or C- segment of the Cas enzyme,

[0029] optionally further comprising a nuclear localization signal,

[0030] optionally further comprising a marker for detection, e.g., a peptide epitope, a fluorescent protein.

[0031] In one or more embodiments, there is a linker, e.g., XTEN80, between each domain or component of the fusion protein.

[0032] In one or more embodiments, the fusion protein comprises, in order from N- to C- terminus:

[0033] dCas9, a KRAB domain,

[0034] dCas9, DNMT3A, DNMT3L,

[0035] KRAB domain, dCas9, DNMT3A, DNMT3L, or

[0036] dCas9, DNMT3A, DNMT3L, KRAB domain.

[0037] The present application also provides use of an agent for inhibiting BRCA1 gene expression and an anticancer drug in the manufacture of a medicament for treating cancer, wherein the agent for inhibiting BRCA1 gene expression, the anticancer drug, and the cancer are as described in any one of the embodiments of the first aspect of the present application.

[0038] The present application also provides a method for treating cancer, comprising administering a therapeutically effective amount of an agent for inhibiting BRCA1 gene expression and a therapeutically effective amount of an anticancer drug, wherein the agent for inhibiting BRCA1 gene expression, the anticancer drug, and the cancer are as described in any one of the embodiments of the first aspect of the present application.

[0039] In one or more embodiments, the anticancer drug is administered at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days after the administration of the agent for inhibiting BRCA1 gene expression.

[0040] The present application also provides a non-therapeutic method for reducing BRCA1 expression in a cell in vitro, comprising increasing the methylation level of a BRCA1 promoter region or a fragment thereof.

[0041] In one or more embodiments, the BRCA1 promoter region is a region from 2000 bp upstream to 1000 bp downstream of the transcription start site.

[0042] In one or more embodiments, the BRCA1 promoter region is from position 41276500 to position 41279500 of human chromosome 17, and the fragment of the BRCA1 promoter region is a fragment comprising position 41277060 to position 41277444 of human chromosome 17 from position 41276500 to position 41279500 of human chromosome 17.

[0043] In one or more embodiments, the BRCA1 promoter region is as shown in SEQ ID NO: 11.

[0044] In one or more embodiments, increasing the methylation level of the BRCA1 promoter region or a fragment thereof refers to increasing the average methylation level of any one or more or all of the CG dinucleotides in the sequence from position 41277060 to position 41277444 of human chromosome 17.

[0045] In one or more embodiments, the method comprises:

[0046] introducing into a cell (1) an sgRNA or a complement thereof, or a nucleic acid construct comprising the sgRNA or the complement thereof, and (2) an epigenetic editing reagent; or

[0047] introducing into a cell comprising an epigenetic editing reagent an sgRNA or a complement thereof, or a nucleic acid construct comprising the sgRNA or the complement thereof,

[0048] The epigenetic reagent is as described in any one of the embodiments of the first aspect.

[0049] The present application also provides a kit or composition comprising (1) an sgRNA or a complement thereof, or a nucleic acid construct comprising the sgRNA or the complement thereof, (2) an epigenetic editing reagent.

[0050] In one or more embodiments, the nucleic acid construct is a cloning vector or an expression vector of the sgRNA or the complement thereof.

[0051] In one or more embodiments, the sgRNA has a sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 2 or a variant thereof having at least 80% sequence identity thereto.

[0052] In one or more embodiments, the epigenetic editing reagent comprises a DNA methyltransferase or an expression vector thereof, and / or a Cas enzyme or an expression vector thereof.

[0053] 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, for example DNMT3L. Preferably, the N- or C- segment of the DNA methyltransferase is fused with DNMT3L.

[0054] In one or more embodiments, the Cas enzyme is Cas9, preferably a Cas9 mutant dCas9 without endonuclease activity. Preferably, the N- or C- segment of the Cas enzyme is fused with a KRAB domain.

[0055] In one or more embodiments, the epigenetic editing reagent further comprises a marker or an expression vector thereof for detection, for example a peptide chain epitope, a fluorescent protein.

[0056] In one or more embodiments, the epigenetic editing reagent comprises a fusion protein or an expression vector thereof, the fusion protein comprising

[0057] (1) a DNA methyltransferase, such as DNMT1, DNMT2, DNMT3A, or DNMT3B,

[0058] (2) a Cas enzyme, such as Cas9, preferably a Cas9 mutant dCas9 without endonuclease activity,

[0059] optionally further comprising a DNA binding protein located at the N- or C- segment of the DNA methyltransferase, such as DNMT3L,

[0060] optionally further comprising a KRAB domain located at the N- or C- segment of the Cas enzyme,

[0061] optionally further comprising a nuclear localization signal,

[0062] optionally further comprising a marker for detection, such as a peptide tag, a fluorescent protein.

[0063] In one or more embodiments, there is a linker, such as XTEN80, between each domain or component of the fusion protein.

[0064] In one or more embodiments, the fusion protein comprises, in order from N- to C- terminus:

[0065] dCas9, a KRAB domain,

[0066] dCas9, DNMT3A, DNMT3L,

[0067] a KRAB domain, dCas9, DNMT3A, DNMT3L, or

[0068] dCas9, DNMT3A, DNMT3L, a KRAB domain.

[0069] In one or more embodiments, the epigenetic editing reagent is a CRISPRoff system or a CRISPR-dCas9-DNMT3A. BRIEF DESCRIPTION OF DRAWINGS

[0070] Figure 1, Design and validation of BRCA1 promoter epigenetic editor. A: Schematic diagram of epigenetic editing strategy applied to BRCA1 promoter. B, C and D: DNA methylation changes within 400 bp range around sgRNA binding site 48 hours after transfection in OC cells A2780 (B), OVCAR3 SKOV3 cells (C) and 293T cells (D). The abscissa is the chromosomal location of the CG site. E and F: Changes in CG methylation level in the BRCA1 promoter region (chromosome 17, locus 4122389) in A2780 cell lines (E) and 293 cell lines (F) as the number of cell culture days increases. G: Expression level of BRCA1 gene in A2780 cells after promoter epigenetic editing. The abscissa represents the number of days of cell culture after editing. H: H3K27Ac modification level of BRCA1 promoter after editing. The abscissa represents the number of days of cell culture after epigenetic editing. Statistical analysis used two-tailed Student's t-test (*P < 0.05).

[0071] Figure 2 , Epigenetic editing of BRCA1 promoter affects the HR repair capacity of OC cells and enhances their sensitivity to drugs. A and B: Double immunofluorescence staining of RAD51 / γH2AX in A2780 (A) or SKOV3 (B) cells exposed to 1 μg / ml DDP. Scale bar, 10 μm. C and D: In A2780 (C) or SKOV3 (D) cells, the number of γH2AX and RAD51 foci in a maximum of 100 cells per group was counted. E: Proliferation curve of A2780 cells after 48 hours of epigenetic editing, expressed as OD450 after 5 days of treatment with 1 μg / ml cisplatin or 10 μM Olaparib. F: Cell number after 5 days of continuous culture, expressed as mean ± SEM. G and H: Apoptosis detection using flow cytometry after annexin V-FITC / propidium iodide (PI) staining. Representative scatter plots of PI (y-axis) versus annexin V (x-axis) are shown (G), as well as the percentage of viable cells, early apoptotic cells, late apoptotic cells and all apoptotic cells (H). Data are the mean ± SEM of triplicate experiments (*P < 0.05, **P < 0.01).

[0072] Figure 3, methylation of CG sites in the BRCA1 promoter is correlated with gene expression levels. A: Schematic diagram of the BRCA1 promoter region. The first exon is represented by a gray rectangle, and the transcription direction is represented by an arrow. The CpG site is represented by a gray thin line, and the rectangle below the thin line represents the CpG island. The rectangle below the CpG island represents the bisulfite amplified fragment sequencing (BSAS) amplified and sequenced region. B: Correlation between the methylation levels of 8 CG sites in the BRCA1 promoter and the BRCA1 expression level shown in the data of the Cancer Genome Atlas (TCGA) Ovarian Cancer (OC) database. The x-axis represents the methylation level of each CG site in the sample, and the y-axis represents the RNA-Seq expectation maximization (RSEM) value of the gene.

[0073] Figure 4 , BRCA1 promoter methylation and its impact on OC development and homologous recombination deficiency (HRD).

[0074] A, B, C, and D: BSAS analysis of BRCA1 promoter methylation levels at CG sites in the promoter region proximal to the transcription start site (TSS) in OC and normal ovarian tissue (A), as well as in HRD OC samples (HRD score ≥42) and HRD-negative samples (HRD score <42) (C); and methylation levels at CG sites in the promoter region upstream of the TSS in OC and normal ovarian tissue (B), as well as in HRD OC samples (HRD score ≥42) and HRD-negative samples (HRD score <42) (D). E and F: Bar chart (E) and scatter plot (F) show the association between DNA methylation levels at Chr17 41277444 in the BRCA1 promoter, BRCA1 mutations, and HRD scores in OC samples. G and H: Bar chart (G) and scatter plot (H) show the association between methylation levels at cg19088651, BRCA1 mutations, and HRD scores in TCGA OC samples. Statistical analysis was performed using a two-tailed Student's t-test. Significance levels are indicated by *P < 0.05, **P < 0.01, and ****P < 0.0001. I: Kaplan-Meier survival analysis was performed to investigate the effects of hypermethylation (indicated by blue) and hypomethylation (indicated by red) of cg19088651 in TCGA OC samples. Survival rates are expressed as days after surgery, and significance was determined at p < 0.05. DETAILED DESCRIPTION

[0075] After in-depth research, the inventors designed an editing method targeting a specific BRCA1 promoter region. Verification of the editing effect revealed that epigenetic inactivation of BRCA1 in OC cells significantly increased sensitivity to platinum and PARP inhibitors.

[0076] The present application provides a kit or composition comprising (1) sgRNA or a complementary sequence thereof, or a nucleic acid construct comprising the sgRNA or the complementary sequence thereof, (2) an epigenetic editing reagent. The kit or composition can increase the methylation level of the region from 41276500 to 41279500, or from 41277060 to 41277444 of the BRCA1 promoter TSS region of a cell.

[0077] In this application, the "TSS region" refers to the transcription start site of a gene, which usually comprises the promoter of the gene, regulatory elements, and transcription factor binding sites, etc. The BRCA1 in this application is located at 17q21.31, and the TSS is located at 41,277,500 (Hg19) in reverse. The epigenetic editing is performed on the DNA region or fragment thereof from 2,000 bp upstream to 1,000 bp downstream of the BRCA1 promoter region (41,276,500-41,279,500). The fragment of the BRCA1 promoter region is a fragment comprising 41277060 to 41277444 of human chromosome 17 from 41276500 to 41279500. In one or more embodiments, the BRCA1 promoter region is as shown in SEQ ID NO: 11, which corresponds to 41279500 to 41276501 of chromosome 17. The chromosome number in this application refers to the human reference genome GRCh37.

[0078] In one or more embodiments, increasing the methylation level of the BRCA1 promoter region or the fragment thereof refers to increasing the average methylation level of any one or more or all of the CG dinucleotides in the sequence from 41277060 to 41277444 of human chromosome 17. The CG dinucleotides from 41277060 to 41277444 of human chromosome 17 include the following positions: 41277060, 41277074, 41277106, 41277115, 41277117, 41277153, 41277160, 41277174, 41277214, 41277232, 41277322, 41277364, 41277381, 41277389, 41277392, 41277394, 41277400, 41277389, 41277392, 41277394, 41277400, 41277426, 41277428, 41277436, 41277444.

[0079] In one or more embodiments, increasing the methylation level of the BRCA1 promoter region or a fragment thereof refers to increasing the methylation level of the following sites in positions 41277060 to 41277444 of human chromosome 17: 41277060, 41277074, 41277106, 41277115, 41277117, 41277153, 41277160, 41277174, 41277214, 41277232, 41277364, 41277381, 41277389, 41277392, 41277394, 41277400, 41277389, 41277392, 41277394, 41277400, 41277426, 41277428, 41277436, 41277444.

[0080] In one or more embodiments, increasing the methylation level of the BRCA1 promoter region or a fragment thereof refers to increasing the methylation level of the following sites in positions 41277060 to 41277444 of human chromosome 17: 41277060, 41277074, 41277106, 41277115, 41277117, 41277153, 41277160, 41277174, 41277214, 41277232, 41277322, 41277364, 41277381, 41277389, 41277392, 41277394, 41277400, 41277389, 41277392, 41277394, 41277400, 41277428, 41277436, 41277444.

[0081] In one or more embodiments, increasing the methylation level of the BRCA1 promoter region or a fragment thereof refers to increasing the methylation level of the following sites in positions 41277060 to 41277444 of human chromosome 17: 41277060, 41277074, 41277106, 41277115, 41277117, 41277153, 41277160, 41277174, 41277214, 41277232, 41277322, 41277364, 41277381, 41277389, 41277392, 41277394, 41277400, 41277389, 41277392, 41277394, 41277400, 41277426, 41277428, 41277436, 41277444.

[0082] In one or more embodiments, increasing the methylation level of the BRCA1 promoter region or a fragment thereof refers to increasing the methylation level of the following sites in positions 41277060 to 41277444 of human chromosome 17: 41277060, 41277074, 41277106, 41277115, 41277117, 41277153, 41277160, 41277174, 41277214, 41277232, 41277364, 41277381, 41277389, 41277392, 41277394, 41277400, 41277389, 41277392, 41277394, 41277400, 41277428, 41277444.

[0083] In one or more embodiments, increasing the methylation level of the BRCA1 promoter region or a fragment thereof refers to increasing the methylation level of the following sites in positions 41277060 to 41277444 of human chromosome 17: 41277060, 41277074, 41277106, 41277115, 41277117, 41277153, 41277160, 41277174, 41277214, 41277232, 41277364, 41277381, 41277389, 41277392, 41277394, 41277400, 41277389, 41277392, 41277394, 41277400, 41277428, 41277436, 41277444.

[0084] In one or more embodiments, increasing the methylation level of the BRCA1 promoter region or a fragment thereof refers to increasing the methylation level of the following sites in positions 41277060 to 41277444 of human chromosome 17: 41277060, 41277074, 41277106, 41277115, 41277117, 41277153, 41277160, 41277174, 41277214, 41277232, 41277322, 41277364, 41277381, 41277389, 41277392, 41277394, 41277400, 41277389, 41277392, 41277394, 41277400, 41277428, 41277444.

[0085] In one or more embodiments, increasing the methylation level of the BRCA1 promoter region or a fragment thereof refers to increasing the methylation level of the following sites in human chromosome 17 41277060 to 41277444: 41277060, 41277074, 41277106, 41277115, 41277117, 41277153, 41277160, 41277174, 41277214, 41277232, 41277364, 41277381, 41277389, 41277392, 41277394, 41277400, 41277389, 41277392, 41277394, 41277400, 41277428, 41277444.

[0086] As used herein, "DNA methylation" or "methylation" can be used interchangeably to refer to a process of obtaining a methyl (CH3) group in a covalent manner, which is a chemical modification process of S-adenosyl methionine (SAM) as a CH3 donor under the catalysis of DNMT at a specific base of a DNA sequence. In this process, cytosine (C) is the most common base for methylation, and C site methylation mainly occurs in CpG sequences. In mammals, some CpG dinucleotides are dispersed in the genome, while others are in dense clusters of CpG islands. CpG islands are usually unmethylated and often located in the promoter region of genes that regulate transcription initiation and inhibition. High methylation of CpG sites in enhancers or promoters usually leads to transcriptional silencing, while low methylation of CpG sites in the genome usually leads to activation of gene expression, so methylation usually regulates gene expression through gene transcription. In the present application, the inventors found that by methylation of a specific region of the promoter of BRCA1 using an epigenetic editing tool, BRCA1 can be inactivated, thereby improving the drug sensitivity of ovarian cancer.

[0087] The DNA methylation ratio is the ratio of methylated cytosine to cytosine, and the representation method is percentage, from 0% to 100%.

[0088] As used herein, the calculation of "methylation level" can be achieved by various means known in the art. Exemplarily herein, the methylation level can be the proportion of the number of cells in which methylation occurs at a site to the total number of cells. The methylation level of multiple sites can be the average, weighted average, median, or obtained by a methylation level calculation program known in the art.

[0089] As used herein, the epigenetic editing tool or epigenetic editing agent can include a CRISPRoff system and sgRNA.

[0090] The CRISPRoff system comprises DNA methyltransferases, which are responsible for adding methyl groups to cytosines on DNA molecules, including DNMT1, DNMT2, DNMT3A, DNMT3B. The CRISPRoff system can also include DNMT3L, which forms a complex by interacting with the DNMT3A protein, and can enhance the methylation activity of DNMT3A on DNA. The DNA methyltransferases can be fused with a nuclear localization signal for bringing the fusion protein into the nucleus.

[0091] The CRISPRoff system also comprises a Cas enzyme, such as dCas9 without endonuclease activity. The Cas enzyme and sgRNA bind to recognize a specific location. The CRISPRoff system can also include a domain KRAB for continuing to recruit DNA methyltransferases to increase the level of DNA methylation and recruit histone deacetylases to reduce the acetylation of H3. The Cas enzyme can be fused with a nuclear localization signal for bringing the fusion protein into the nucleus

[0092] The CRISPRoff system can also comprise a peptide tag for antibody detection, such as HA.

[0093] The CRISPRoff system can also comprise a fluorescent protein for observing the expression of the fusion protein, preferably the fluorescent protein is BFP.

[0094] The CRISPRoff system can be a fusion protein comprising the components or an expression vector thereof. For example, the fusion protein comprises (1) a DNA methyltransferase, such as DNMT1, DNMT2, DNMT3A, or DNMT3B, and (2) a Cas enzyme, such as Cas9, preferably a Cas9 mutant dCas9 without endonuclease activity. The fusion protein optionally further comprises a DNA binding protein, such as DNMT3L, located at the N- or C- segment of the DNA methyltransferase; optionally further comprises a KRAB domain located at the N- or C- segment of the Cas enzyme; optionally further comprises a nuclear localization signal; optionally further comprises a marker for detection, such as a peptide tag, a fluorescent protein. There can or can not be a linker, such as XTEN80, between the domains or components of the fusion protein.

[0095] In one or more embodiments, the CRISPRoff system comprises a fusion protein or an expression vector thereof, which comprises, from N- to C-terminus: DNMT3A, DNMT3L, XTEN80, dCas9, HA, NLS, BFP, KRAB. An exemplary CRISPRoff plasmid is available from Addgene.

[0096] sgRNA is a guide RNA, which is an important component in the CRISPR gene knockout and knock-in system, and has two parts: tracRNA and crRNA. After fusion expression, sgRNA can well perform the function of guide, combine with Cas9 protein, and guide Cas9 enzyme to target genomic DNA for cleavage. In some embodiments, the sgRNA herein has the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0097] The present application also provides use of a substance (including sgRNA, epigenetic editing reagent) for increasing the methylation level of the region 41276500-41279500 or the region from 41277060 to 41277444 of the BRCA1 promoter of a cell in the preparation of a drug for treating ovarian cancer or for increasing the sensitivity of ovarian cancer cells to platinum drugs and / or PARP inhibitors. The BRCA1 expression of the ovarian cancer is up-regulated or has no significant change compared with a control, and the BRCA1 expression of the tumor cells is up-regulated or has no significant change compared with a control. The control can be the BRCA1 expression of normal cells or tissues of the subject, the BRCA1 expression of cells adjacent to the cancer, the average BRCA1 expression of a population, and the like negative controls commonly used in the art. The ovarian cancer cells include SKOV3 cells or A2780 cells. In the drug, the sgRNA, the epigenetic editing reagent, the platinum drug, and the PARP inhibitor are all active ingredients.

[0098] The "platinum drug" mainly refers to a drug for treating neoplastic diseases, including cisplatin, carboplatin, nedaplatin, oxaliplatin, and lobaplatin. In some embodiments, the platinum drug is cisplatin. The "PARP inhibitor" is also called poly (ADP-ribose) polymerase inhibitor, which can affect the self-replication mode of cancer cells, including olaparib, niraparib, talazoparib, and pamiparib. In some embodiments, the PARP inhibitor is olaparib.

[0099] In addition to these active ingredients, the medicament also comprises pharmaceutically acceptable excipients. The term "pharmaceutically acceptable excipient" refers to a carrier and / or excipient with which the subject and the active ingredients are compatible in a pharmacological and / or physiological sense that is well-known in the art (see, e.g., 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 dosages and concentrations employed. Such excipients include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof. In certain embodiments, the medicament can contain substances to improve, maintain or preserve, for example, the pH, osmolarity, 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 medicament can be determined depending on the intended route of administration, mode of delivery, and desired dosage.

[0100] The medicament for in vivo administration is typically provided in the form of a sterile preparation. Sterilization is accomplished by filtration through a sterile filtration membrane. This method can be used for sterilization before or after lyophilization and rehydration of the composition. The medicament of the present application can be selected for parenteral delivery. The medicament for parenteral administration can be stored in lyophilized form or in solution. It is prepared by conventional methods, for example, with physiological saline or aqueous solutions containing dextrose and other auxiliary agents. The parenteral medicament is usually placed in a container with a sterile access hole, such as an intravenous solution bag or a vial with a plug that can be pierced by a hypodermic needle. Alternatively, the medicament can be selected for inhalation or delivery through the digestive tract, such as orally. The preparation of the pharmaceutically acceptable medicament is within the skill in the art. Other medicaments will be apparent to those skilled in the art. Techniques for formulating a variety of other sustained or controlled delivery means, such as liposome carriers, bioerodible microparticles or porous beads, and depot injections, are also known to those skilled in the art.

[0101] Once formulated, the medicament is stored in a sterile vial in the form of a solution, suspension, gel, emulsion, solid, crystal, or in dehydrated or lyophilized powder. The formulation can be stored in ready-to-use form or in a form that is rehydrated before administration (e.g., lyophilized).

[0102] The present application also provides a method of treating cancer, comprising administering a therapeutically effective amount of an agent that inhibits BRCA1 gene expression and a therapeutically effective amount of an anticancer drug. Illustratively, the agent that inhibits BRCA1 gene expression is an agent that increases the methylation level of the BRCA1 promoter region or a fragment thereof described herein, such as sgRNA and optional CRISPR off system. The agent that inhibits BRCA1 gene expression and the anticancer drug can be administered sequentially, separately, or simultaneously. The separate administration refers to the administration of the anticancer drug at intervals from the administration of the agent that inhibits BRCA1 gene expression or the administration of the agent that inhibits BRCA1 gene expression at intervals from the administration of the anticancer drug. In one or more embodiments, the anticancer drug is administered at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days after the administration of the agent that inhibits BRCA1 gene expression.

[0103] "Treating" refers to the use of a therapeutic regimen described herein to a subject to achieve at least one positive therapeutic effect (such as, a reduction in the number of cancer cells, a reduction in tumor size, a reduction in the rate of cancer cell infiltration into peripheral organs, or a reduction in the rate of tumor metastasis or tumor growth). The therapeutic regimen effective to treat a patient can vary depending on a variety of factors such as the patient's disease state, age, body weight, and the ability of the therapy to elicit an anti-cancer response in the subject. The therapeutically effective amount of the agent that increases the methylation level of the BRCA1 promoter region to be employed will depend, for example, on the therapeutic effect desired and the target. Those of skill in the art will appreciate that appropriate dosage levels for treatment will vary depending on the molecule delivered, the indication, the route of administration, and the size (body weight, body surface or organ size) and / or condition (age and general health) of the patient. In some embodiments, the clinician can titer the dosage and route of administration to optimize the therapeutic outcome. For example, from about 10 micrograms per kilogram body weight to about 50 milligrams per kilogram body weight per day.

[0104] The frequency of dosing will depend on the pharmacokinetic parameters of the agent used in the formulation. The clinician typically will administer the pharmaceutical composition containing the agent until a dosage is reached that achieves the desired effect. The pharmaceutical composition can therefore be administered as a single dose, multiple doses (which can or can not contain the same amount of the desired molecule), or as a continuous infusion over time through an implanted device or catheter.

[0105] The route of administration of the pharmaceutical composition is according to known methods, for example, by oral, by injection via intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, portal, or intralesional routes; by sustained release systems, or by implanted devices.

[0106] The present application also provides a non-therapeutic method of reducing BRCA1 expression in a cell in vitro, comprising an agent that increases the methylation level of the BRCA1 promoter region or a fragment thereof.

[0107] The present application also provides a method and reagent for increasing the sensitivity of ovarian cancer cells to platinum drugs and PARP inhibitors. The method mainly changes the response of ovarian cancer cells to platinum drugs by epigenetic editing of the BRCA1 promoter. This editing can be achieved by using a specific epigenetic editing tool, CRISPRoff.

[0108] Specifically, the method of the present application comprises the following steps: 1. Design and synthesize the epigenetic editing tool CRISPRoff. 2. Design and synthesize the guide RNA for guiding the tool to edit the BRCA1 promoter specifically. 3. Introduce the tool and guide RNA into ovarian cancer cells to edit the BRCA1 promoter. 4. Verify the editing effect by experimental methods, and whether the sensitivity of the edited ovarian cancer cells to platinum drugs has increased.

[0109] In addition, the present application also provides a reagent for implementing the above-mentioned method. The reagent includes the tool CRISPRoff for epigenetic editing, and the guide RNA (gRNA) for guiding the tool to edit the BRCA1 promoter. Through the method and reagent of the present application, the sensitivity of ovarian cancer cells to platinum drugs and PARP inhibitors can be effectively increased, thereby improving the effect of drug treatment of ovarian cancer.

[0110] Embodiments

[0111] Materials and methods

[0112] 1. DNA methylation analysis of OC samples in the data set

[0113] Analysis of DNA methylation and gene expression data within the BRCA1 promoter in OC tumor samples utilized public datasets in The Cancer Genome Atlas (TCGA). Correlation analysis of BRCA1 promoter methylation and gene expression in 398 TCGA ovarian serous cystadenocarcinoma samples was performed by cBioPortal (https: / / www.cbioportal.org / ). The promoter region was defined as the region from 2000 bp upstream to 1000 bp downstream of the transcription start site (TSS). DNA methylation data, SNP array information, and survival data of 602 OC samples were obtained from TCGA (https: / / portal.gdc.cancer.gov). Kaplan-Meier (K-M) method was used to estimate the survival rate associated with DNA methylation using the survminer v0.4.9 R package. This package uses log-rank test to compare survival time, with a significance level of p<0.05.

[0114] 2. Plasmid construction

[0115] The plasmid CRISPRoff was purchased from Addgene (Addgene #167981). The sgRNA plasmid was generated by restriction cloning the protospacer downstream of the U6 promoter using the BsmBI cleavage site in the pLentiGuide-puro plasmid. It is worth noting that the CRISPRoff plasmid also expresses a blue fluorescent protein (BFP) marker to facilitate measurement of transfection efficiency. The sgRNA sequences against the BRCA1 promoter are as follows: (SEQ ID NO: 1) 5'-TGAAGGCCTCCTGAGCGCAG-3' against chromosome 17 coordinate 41277324, (SEQ ID NO: 2) 5'-TTACCCAGAGCAGAGGGTGA-3' against chromosome 17 coordinate 41277307.

[0116] 3. Cell culture and transfection

[0117] Three human cell lines, 293T, A2780 and SKOV3, were cultured. 293T cells were cultured in Dulbecco's Modified Eagle Medium (DMEM), while the OC cell lines A2780 and SKOV3 were cultured in RPMI-1640 medium. These cell cultures were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (P / S) antibiotics. The culture conditions included maintaining the cells at a temperature of 37°C and ensuring proper humidity in an environment containing 5% CO2. When 293T, A2780 and SKOV3 cells reached 70-80% confluency, transfection experiments were performed using 300 ng of CRISPRoff plasmid and 150 ng of sgRNA-encoding plasmid in combination with Lipofectamine 3000 (Thermo Fisher Scientific, USA) in 24-well plate Opti-MEM medium.

[0118] 4. Clinical sample collection

[0119] For validation analysis, 81 OC formalin-fixed paraffin-embedded (FFPE) samples were collected from Xiangya Hospital, Central South University (Changsha, China) from January 2017 to December 2020. The average age of the patients at diagnosis was (50.6 ± 11.0) years. Among them, we obtained 46 high-grade serous ovarian cancer (HGSOC) samples, as well as matched peripheral blood samples for HRD analysis. Our study obtained ethical approval from the local committee, and all participants provided written informed consent.

[0120] 5. HRD score calculation and BRCA1 / 2 gene mutation analysis

[0121] The genomic scar signature was used to calculate the HRD score using the R program as previously described [Marquard AM, Eklund AC, Joshi T, et al. Biomark Res. 2015;3:9. doi:10.1186 / s40364-015-0033-4]. This calculation included determining telomeric allelic imbalance (TAI), large-scale mutation (LST), and loss of heterozygosity (LOH) scores. TAI indicates that the region of allelic imbalance extends to the telomere, LOH indicates that the chromosomal LOH region is longer than 15 Mb, and LST refers to the breakpoints between regions longer than 10 Mb after filtering out regions shorter than 3 Mb. The HRD score was calculated as the sum of the TAI, LST, and LOH scores. In the TCGA cohort, the HRD score was calculated using SNP microarray data (Affymetrix GenomeWideSNP6 array). Both tumor and corresponding normal tissues were analyzed. The analytical pipeline used for next-generation sequencing (NGS) was also adapted to analyze clinical tumor samples. The 3DMed Onco GPS Tissue NGS kit (3DMed, China) used targets 733 tumor-related genes, including BRCA1 / 2. Probes were used to capture the target genes and then sequenced on the HiSeq platform (Illumina, Inc.). BRCA1 / 2 gene mutations were also evaluated using this gene panel. In brief, the raw sequencing data were aligned with the reference human genome (February 2009, GRCh37 / hg19) using BWA software (version 0.5.9) and SAMtools (version 0.1.18). The Genome Analysis Toolkit (version 3.5) was used for variant calling, using parameters such as UnifiedGenotyper, HaplotypeCaller, and VariantFiltration. Subsequently, ANNOVAR was used for variant annotation. The interpretation of the variants followed the American College of Medical Genetics and Genomics (ACMG) guidelines. Pathogenic or likely pathogenic variants include frameshift insertions or deletions (indels), nonsense mutations, uncorrected splice site variants, large deletions, and missense mutations that impair protein function as determined by functional testing. Only variants that are pathogenic or likely pathogenic are classified as BRCA1 / 2 variants.

[0122] 6. Bisulfite Amplified Sequencing (BSAS)

[0123] DNA from cell line samples and FFPE samples was extracted using High Pure PCR Template Preparation Kit (Roche, USA) and GeneRead DNA FFPE Kit (QIAGEN, Germany), respectively. The DNA methylation level of candidate genes was evaluated by bisulfite amplification and next-generation sequencing (NGS). Specifically, 10 nanograms of genomic DNA was bisulfite-converted with EZ DNA Methylation-Lightning Kit (Zymo Research, USA). Subsequently, we performed PCR amplification of the target sequence, and constructed a second-generation sequencing library using TaKaRa EX Taq Hot Start Version Kit (China). The amplification primers were as follows: BRCA1 forward primer: (SEQ ID NO: 3) 5'-GGAAAGGGATAGGGGGTTTAAGTGATGTT-3', (SEQ ID NO: 4) BRCA1 reverse primer: 5'-AATTCACAARGCCTTARGCCTCTCAAATTC-3'. The library was sequenced using the NGS HiSeq platform (Illumina, Inc.) for 2X150bp. The quality of the sequencing data was verified using the FastQC software package. Adapter removal and quality trimming were performed using Cutadapt v1.9.1, and the reads were aligned to the reference genome (UCSC hg19) using Bismark. The methylation level of individual cytosines was determined using the R package MethylKit v1.26.0.

[0124] 7. Quantitative real-time polymerase chain reaction

[0125] Total RNA was extracted from cultured cells and tissue samples using TRIzol Reagent (SIGMA, USA) according to the manufacturer's instructions. The CFX96 Real-Time PCR quantitative PCR instrument (Bio-Rad, USA) and SYBR Green PCR mix (TaKaRa, China) were used for qPCR reaction. The comparative Cq method was used to calculate the fold change. The amplification conditions were 60°C for 60 minutes, followed by 95°C for 5 minutes, 95°C for 15 seconds, 60°C for 1 minute, and 45 PCR cycles. BRCA1 primers (forward 5'-ACAGCTGTGTGGTGGCTTCTGTG-3' (SEQ ID NO: 5), reverse 5'-CATTGTCCTCTGTCCAGGCATC-3' (SEQ ID NO: 6)), GAPDH primers (forward 5'-TCATTGACCTCAACTACATGGTTT-3' (SEQ ID NO: 7), reverse 5'-GAAGATGGTGATGGGATTTC-3' (SEQ ID NO: 8)).

[0126] 8. Chromatin immunoprecipitation (ChIP)-qPCR

[0127] A2780 cells were briefly frozen in 125 mM glycine for 5 min at room temperature and then fixed in 1% formaldehyde for 10 min. After fixation, the cells were centrifuged at 1,000 g for 10 min at 4 °C and washed with PBS three times. Subsequently, the cell pellets were resuspended in 10 mL ChIP lysis buffer (containing 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Igepal, and protease inhibitors) and incubated on a 4 °C rotary shaker for 60 min. The nuclei were then separated by centrifugation (10 min, 4 °C, 1,000 g), resuspended with SDS lysis buffer (containing 1% SDS, 10 mM EDTA, and 50 mM Tris-HCl, pH 8.0), and incubated on ice for 30 min. The SDS was diluted with lx TE, and sonication was performed using a Covaris M220 (Covaris, USA). The efficiency of sonication was evaluated by agarose gel electrophoresis. Subsequently, ChIP was performed using 2 pg of H3K27ac antibody (A7253, ABclonal, China) at 4 °C for 16 h. The immune complexes were captured with 20 pl of magnetic protein A / G beads (Thermo Fisher Scientific, USA) at 4 °C for 2 h. The beads were washed twice with RIPA, three times with ChIP wash buffer (containing 100 mM Tris pH 8, 500 mM LiCl, and 1% deoxycholic acid), and once with ChIP wash buffer supplemented with 150 mM NaCl. The ChIP complexes were eluted in 100 pl of ChIP elution buffer (containing 50 mM NaHCO3 and 1% SDS) for 30 min and reverse-crosslinked at 65 °C overnight in the presence of 0.5 M NaCl. The ChIP DNA was purified using a QIAquick PCR purification kit (QIAGEN, Germany). Quantitative PCR was performed on ChIP and input control samples using SYBR Premix Ex Taq™ (TaKaRa, China) and a CFX96 Real-Time PCR detector (Bio-Rad, USA) with SYBR Green PCR Mix (TaKaRa, China). The ChIP amplification primers for the BRCA1 promoter were as follows: (SEQ ID NO: 9) 5'-CGTATTCTGAGAGGCTGCTG-3' and (SEQ ID NO: 10) 5'-GGAAGTCTCAGCGAGCTCAC-3'. The ChIP enrichment relative to the input sample was determined using the dCq method (dCq = Cq[ChIP] - Cq[input]).

[0128] 9. Cell proliferation assay

[0129] A2780 cells were seeded into 6-well plates 24 hours before transfection to reach 70-90% confluence. Methylation editing plasmid pLentiGuide-puro-BRCA1 and CRISPRoff (500 ng each) were co-transfected using Lipofectamine 3000 (Thermo Fisher Scientific, USA). Non-editing plasmid was used as a control. 24 hours after transfection, cells were collected and seeded into 96-well plates at a density of 2 x 104 per well. After 24 hours of culture, different concentrations of cisplatin (DDP) (0.75 pg / ml and 1 pg / ml) (SIGMA, USA) and olaparib (10 mM and 20 mM) (MedChemExpress, USA) were added for drug sensitivity experiments. Cell counting kit-8 (China DOJINDO) was added at day 0, 1, 3, 4 and 7 after drug treatment. After 2 hours of culture, the optical density at 450 nm (OD450) was measured to analyze cell proliferation.

[0130] 10. Apoptosis detection

[0131] After trypsin digestion of adherent cells without EDTA, cells were collected by centrifugation at 300 x g for 5 minutes at 4°C. The apoptosis rate was determined by cell cycle and apoptosis analysis kit (Yeasen, China). Specifically, cells were washed twice with pre-cooled PBS. About 1-5 x 105 cells were collected. The PBS was aspirated, and the cells were resuspended in 100 pL of 1 x binding buffer. Five microliters of Annexin V-FITC and 10 microliters of PI staining solution were added and mixed gently. The mixture was incubated at room temperature for 10-15 minutes in the dark. After adding 400 pL of 1 x binding buffer and mixing thoroughly, the sample was placed on ice and analyzed using a flow cytometer within 1 hour.

[0132] 11. Homologous recombination (HR) detection

[0133] To assess the HR capacity of A2780 and SKOV3 cells, double immunofluorescence staining for γH2AX and RAD51 was performed. Cells were transfected with pLentiGuide-puro-BRCA1 and CRISPRoffv2.1 or a control plasmid. After 5 days of culture, cells were exposed to 10 μM DDP (SIGMA, USA) for 24 hours. Subsequently, cell nuclei were extracted on ice for 10 minutes using a buffer containing 20 mM HEPES (pH 7.4), 20 mM NaCl, 5 mM MgCl2, 0.5% NP-40, 1 mM DTT, and protease inhibitors. Cells were then rinsed with ice-cold PBS and fixed with 4% PFA for 10 minutes at room temperature. Cells were then blocked for 30 minutes in immunofluorescence (IF) blocking buffer consisting of 10% goat serum, 0.5% NP-40, and 0.5% saponin in PBS. Cells were then incubated with primary antibodies diluted in blocking buffer: mouse anti-RAD51 (GeneTex, USA) and rabbit anti-γ-H2AX (Cell Signaling, USA) for 2 hours at room temperature. After primary antibody incubation, cells were rinsed with PBS (three times for 5 minutes) and then stained with fluorescent secondary antibodies: goat anti-rabbit IgG (H+L) secondary antibody, DyLight 488 (Thermo Fisher Scientific, USA) and goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 555 (Thermo Fisher Scientific, USA) for 1 hour at room temperature. After secondary antibody staining, cells were washed as described above, mounted with ProLong Gold mounting medium (Thermo Fisher Scientific, USA), and imaged using Ti-E+A1R+STORM10-1 (Nikon, Japan). Data from 100 cells were collected in five to ten fields of view in two independent experiments.

[0134] Example 1: Design and validation of BRCA1 promoter epigenetic editor

[0135] The epigenetic editing plasmid CRISPRoff (DNMT3A-DNMT3L-XTEN80-dCas9-HA-2xNLS-BFP-KRAB) can enhance DNA methylation while reducing histone modifications (such as H3K27ac). The inventors designed two sgRNAs (targeting chromosome 17 coordinates 41277324 and 41277307, respectively), focusing on the BRCA1 TSS region, and then constructed the pLentiGuide-puro plasmid for precise site-specific epigenetic modification ( Figure 1A). CRISPR off plasmid and pLentiGuide-puro plasmid were transfected in 293T and A2780. Subsequently, the expression of fluorescent protein on the plasmid was observed for five days using a fluorescence microscope, aiming to evaluate the expression efficiency and stability. The results clearly showed that the fluorescent protein expression completely disappeared on the fifth day, indicating that the epigenetic editing plasmid presented transient expression in the cells.

[0136] Further study was made on the changes of DNA methylation level after editing. 48 hours after transfection, the DNA methylation level in the range of 400bp upstream and downstream of the sgRNA binding site (position 41277060 to position 41277444 of chromosome 17) in OC cells A2780 and 293T cells increased significantly Figure 1 , B, C and D), these figures represent the DNA methylation level of mixed cells from 0% to 100%, indicating that these cells are edited at different positions or degrees. In A2780 cells, the methylation of all detected CG sites was significantly increased after editing, except for the position 41277322 covered by sgRNA binding; in SKOV3 cells, the methylation of all detected CG sites was significantly increased after editing, except for the position 41277426; in 293T cells, the methylation of all detected CG sites was significantly increased after editing, except for the position 41277322 covered by sgRNA binding and the last position 41277436 of the fragment. The DNA methylation degree of A2780 and 293T cells is higher, and the methylation degree of some CGs is increased by more than 50%. Notably, the DNA methylation of the sgRNA binding site itself changes little. After continuous culture of edited cells, the DNA methylation level gradually decreases; but it is worth noting that in A2780 cells, the DNA methylation level in the TSS region is still significantly higher than that of the control group even on the 28th day Figure 1 , E), while the methylation level of 293T cells continues to increase within 14 days Figure 1 , F). In addition, analysis of BRCA1 gene expression in A2780 cells showed that from the second day to the eighth day after editing, the mRNA level was significantly lower than that of unedited control cells Figure 1 , G). Regarding the histone modification in A2780 cells, we found that the level of H3K27ac modification was significantly lower than that of the control group Figure 1 , H). In summary, these findings clearly confirmed our effect of inhibiting BRCA1 expression in ovarian cancer cells through epigenetic editing.

[0137] Example 2: Epigenetic editing of BRCA1 promoter affects the HR repair ability of OC cells and increases their sensitivity to drugs

[0138] After confirming that significant epigenetic modification changes and gene expression changes occurred in the BRCA1 promoter region after editing, the changes in the HR repair capacity of the cells and their sensitivity to platinum drugs and poly (ADP-ribose) polymerase inhibitors (PARPi) were detected. We first determined the appropriate concentrations of cisplatin and olaparib in A2780 cells and found that through cell proliferation experiments, 1μg / ml cisplatin and 10μM olaparib could inhibit about half of the cell growth after 5 days, which was selected as the subsequent experimental drug concentration (data not shown). The cells were transfected with the BRCA1 epigenetic editing plasmid for 5 days, 1μg / ml cisplatin was added, and the HR capacity was determined by γH2AX / RAD51 double immunofluorescence staining after 1 day of treatment. γH2AX foci represent the location of double-strand breaks on the chromosome, while RAD51 foci represent ongoing HR repair. The ratio of the two co-localizations represents the HR repair capacity. The results showed that the RAD51 foci of drug-treated BRCA1 promoter-edited cells were significantly increased in OC cells A2780 ( Figure 2 ,A) and SKOV3( Figure 2 , B) were significantly reduced. The counting results showed that in A2780 cells, the average number of RAD51 foci in edited A2780 cells was 15.8, while the average number of RAD51 foci in the unedited control group was 18.7 ( Figure 2 , C), while in SKOV3 cells, the average number of foci in unedited and edited cells was 34.5 and 29.3, respectively ( Figure 2 , D). These results indicate that epigenetic inactivation of the BRCA1 promoter reduces HR repair capacity. For drug sensitivity testing, 48 hours after epigenetic editing, A2780 cells were treated with 1 μg / ml cisplatin and 10 μM olaparib for 5 consecutive days. Cell counts showed that cell proliferation was significantly reduced after epigenetic editing of the BRCA1 promoter ( Figure 2 , E). Five days after cisplatin treatment, the number of cells with promoter epigenetic editing was reduced by 32% compared with unedited cells, while that in the olaparib-treated group was reduced by 19% ( Figure 2 , F). The above results indicate that the increase in epigenetic modification in the BRCA1 promoter region inhibits expression, leading to a significant increase in the sensitivity of cells to platinum drugs and PARPi drugs. Interestingly, in the control group without drug treatment, the number of cells in the sgBRCA1 group decreased by 14% on the 5th day, which was also significantly lower than that in the control group ( Figure 2 , F). We analyzed the apoptosis of cells after drug treatment ( Figure 2 , G), Under DDP treatment, the apoptotic rate of A2780 cells with epigenetic editing of the BRCA1 promoter was significantly higher than that of cells without editing ( Figure 2, H). Even in the absence of drug treatment, cells that have undergone epigenetic editing exhibit a higher proportion of apoptosis Figure 2 , H). This result suggests that epigenetic editing of the BRCA1 promoter can increase sensitivity to drugs by promoting apoptosis.

[0139] Example 3, DNA methylation profiling of BRCA1 gene promoter in OC

[0140] BRCA1 is located at 17q21.31, with the TSS at reverse 41,277,500 (Hg19). According to the definition of the promoter, we selected a DNA fragment from 2,000 bp upstream to 1,000 bp downstream of the TSS (41,276,500-41,279,500) for study. Within this promoter region, there are three CpG islands, located at 41,279,075-41,277,986, 41,277,694-41,277,244, and 41,277,206-41,276,698, respectively. The latter two CpG islands are contiguous, forming a region that includes the TSS, part of exon 1, and intron 1. This region, together with the first CpG island, constitutes two CG dense regions, with eight CG dinucleotides having methylation data in the TCGA OC database Figure 3 , A). After analyzing the methylation levels of these CGs in the TCGA OC dataset and their correlation with gene expression, we found that in tumor tissues, the six CGs near the TSS were mainly hypomethylated, with a few showing hypermethylation. In contrast, the two CGs within the CpG island (1000 base pairs upstream of the TSS) were generally hypermethylated, with a few samples showing hypomethylation. The correlation between methylation and expression of the two CGs upstream of the TSS was negligible or weak Figure 3 , B).

[0141] We collected OC tissue samples and performed a comprehensive analysis of DNA methylation levels in two regions of the BRCA1 promoter. We used BSAS detection to amplify and sequence two fragments on chromosome 17 within these two regions: 41,277,787-41,277,627 and 41,275,000-41,277,199 Figure 3 , A). Analysis showed that in normal ovarian tissues, the DNA methylation levels of all eight CGs near the TSS were below 1%. In contrast, in ovarian cancer tissues, some samples showed higher methylation levels, with an average methylation level of 10-15%, indicating a significant difference between the two groups of tissues Figure 4, A). In contrast, the 8 CGs within the CpG island upstream of TSS were highly methylated in both normal ovarian tissues and OC tissues, with no significant difference between the two groups Figure 2 , B). These results were consistent with the results of TCGA samples. It can be inferred that the methylation level of CG near TSS is closely related to the expression level of tumor suppressor gene BRCA1, and the increase of methylation level may be related to the inactivation of BRCA1 promoter in OC. In addition, we also detected the methylation of BRCA1 promoter in clinical cases, which were divided into HRD positive group (HRD score ≥ 42) and HRD negative group (HRD score < 42). Compared with HRD negative samples, the DNA methylation level of 8 CG sites near TSS in HRD positive samples was significantly higher Figure 4 , C). In contrast, the methylation level of 8 CG sites within the CpG island upstream of TSS was similar in different HRD score groups Figure 4 , D). In addition, a significant positive correlation was observed between the methylation level of promoter CG sites and HRD score Figure 4 , F and H). Analysis of survival data of TCGA samples found that high methylation in the region near TSS was associated with higher survival rate Figure 4 , I). These results further suggest that the DNA methylation level in the region near TSS is related to homologous recombination deficiency and may affect survival time through different responses to treatment.

Claims

1. Use of an agent that inhibits BRCA1 gene expression in the preparation of a drug for treating cancer, a drug for improving the efficacy of an anticancer drug, or a drug for increasing the sensitivity of tumor cells to an anticancer drug, wherein the anticancer drug is a chemotherapy drug and / or a PARP inhibitor, and the agent that inhibits BRCA1 gene expression is an agent that increases the methylation level of the BRCA1 promoter region or a fragment thereof. Preferably, The chemotherapy drug is a platinum drug, and / or The BRCA1 promoter region is the region from 2000 bp upstream to 1000 bp downstream of the transcription start site.

2. The use according to claim 1, characterized in that The BRCA1 promoter region is from position 41276500 to position 41279500 of human chromosome 17, and the fragment of the BRCA1 promoter region is from position 41276500 to position 41279500 of human chromosome 17, including position 41277060 to position 41277444. Preferably, increasing the methylation level of the BRCA1 promoter region or a fragment thereof refers to increasing the average methylation level of any one or more or all CG dinucleotides in the sequence from position 41277060 to position 41277444 of human chromosome 17.

3. The use according to claim 1, characterized in that The cancer is ovarian cancer, and the tumor cells are ovarian cancer cells.

4. The use according to claim 1, wherein The reagent for increasing the methylation level of the BRCA1 promoter region or a fragment thereof comprises: (1) sgRNA or its complementary sequence, or (2) a nucleic acid construct containing the sgRNA or its complementary sequence, Preferably, The sgRNA has a sequence as shown in SEQ ID NO: 1 and / or SEQ ID NO: 2, or a variant thereof having at least 80% sequence identity thereto, and / or The reagents also include epigenetic editing reagents.

5. The use according to claim 4, characterized in that The epigenetic editing reagent comprises a DNA methyltransferase or an expression vector thereof, and / or a Cas enzyme or an expression vector thereof.

6. The use according to claim 5, characterized in that The epigenetic editing reagent comprises a fusion protein or an expression vector thereof, wherein the fusion protein comprises: (1) DNA methyltransferases, such as DNMT1, DNMT2, DNMT3A or DNMT3B, (2) Cas enzymes, such as Cas9, Optionally, it further comprises a DNA binding protein located in the N segment or C segment of the DNA methyltransferase, Optionally further comprises a KRAB domain located in the N segment or C segment of the Cas enzyme, Optionally further comprises a nuclear localization signal, Optionally, it further comprises a marker for detection, such as a peptide epitope or a fluorescent protein; Preferably, The fusion protein comprises, from N-terminus to C-terminus: dCas9, KRAB domain, dCas9, DNMT3A, DNMT3L, KRAB domain, dCas9, DNMT3A, DNMT3L, or dCas9, DNMT3A, DNMT3L, KRAB domains.

7. Use of an agent for inhibiting BRCA1 gene expression and an anticancer drug in the preparation of a drug for treating cancer, wherein the agent for inhibiting BRCA1 gene expression, the anticancer drug, and the cancer are as described in any one of claims 1 to 6.

8. A non-therapeutic method for reducing BRCA1 expression in cells in vitro, comprising increasing the methylation level of the BRCA1 promoter region or a fragment thereof, wherein the BRCA1 promoter region is from position 41276500 to position 41279500 of human chromosome 17, and the fragment of the BRCA1 promoter region is from position 41276500 to position 41279500 of human chromosome 17, including positions 41277060 to 41277444. Preferably, increasing the methylation level of the BRCA1 promoter region or a fragment thereof refers to increasing the average methylation level of any one or more or all CG dinucleotides in the sequence from position 41277060 to position 41277444 of human chromosome 17.

9. A kit or composition comprising (1) sgRNA or its complementary sequence, or a nucleic acid construct containing the sgRNA or its complementary sequence, (2) an epigenetic editing agent, Preferably, The nucleic acid construct is a cloning vector or an expression vector of the sgRNA or its complementary sequence, and / or The sgRNA has a sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2, or a variant thereof having at least 80% sequence identity thereto, and / or The epigenetic editing reagent comprises a DNA methyltransferase or an expression vector thereof, and / or a Cas enzyme or an expression vector thereof.

10. The kit or composition according to claim 9, wherein The epigenetic editing reagent comprises a fusion protein or an expression vector thereof, wherein the fusion protein comprises: (1) DNA methyltransferases, such as DNMT1, DNMT2, DNMT3A or DNMT3B, (2) Cas enzymes, such as Cas9, Optionally, it further comprises a DNA binding protein located in the N segment or C segment of the DNA methyltransferase, Optionally further comprises a KRAB domain located in the N segment or C segment of the Cas enzyme, Optionally further comprises a nuclear localization signal, Optionally, it further comprises a marker for detection; Preferably, the fusion protein comprises, from N-terminus to C-terminus: dCas9, KRAB domain, dCas9, DNMT3A, DNMT3L, KRAB domain, dCas9, DNMT3A, DNMT3L, or dCas9, DNMT3A, DNMT3L, KRAB domains.

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