Composition for epigenetic editing and editing and cell selection method
Through the lentiviral vector of Cas functional sequence, TET functional sequence and sgRNA, high-precision genomic methylation editing and cell selection are achieved, solving the problem of dynamic monitoring of methylation characteristics during tumor metastasis, and promoting the development of new gene discovery and treatment strategies.
Patent Information
- Application Number
- CN202510277863.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-10
- Publication Date
- 2025-07-25
AI Technical Summary
The existing technology lacks high-precision screening tools to achieve accurate methylation changes in specific genome sites, which limits the dynamic monitoring of methylation characteristics during tumor metastasis and new gene discovery, and affects the in-depth research of tumor metastasis process and the development of therapeutic strategies.
Using lentiviral vectors including Cas functional sequences, TET functional sequences and sgRNAs targeting target sequence sites, target cell lines are screened through lentiviral infection, cell lines that stably express marker molecules are screened, and proliferation culture and genomic DNA and RNA sequencing are carried out, sgRNA is designed and synthesized for demethylation editing, and cells with the desired phenotype are selected.
The methylation degree of high-precision designated sites of the genome is rewrite, the expression level of target genes is regulated, and the characteristic map of genome methylation changes is drawn through high-throughput screening work, new genes that are silenced due to hypermethylation are discovered, and the methylation pattern of the tumor metastasis process is analyzed, which supports a more comprehensive treatment strategy.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of gene expression regulation, and particularly relates to a composition for epigenetic editing, and also relates to an editing and cell selection method using the composition. Background Art
[0002] DNA methylation is one of the most intensively studied epigenetic modifications at present, and has attracted much attention in the early screening, diagnosis and treatment prediction of tumors. However, the methylation feature transformation during tumor metastasis and its shaping of the tumor microenvironment are still unclear. Current research mainly focuses on analyzing various tumor-specific and common abnormal DNA methylations and converting them into clinical detection kits.
[0003] However, during tumor metastasis and treatment resistance, methylation modification also plays an indispensable role. Traditionally, DNA methylation research and DNA methylation-based biomarker research mainly focus on the hypermethylation effect of CpG islands in the promoters of tumor suppressor genes. However, even within a single promoter region, not all CpG islands are functionally the same. With the in-depth research, researchers found that even the methylation of specific CpG dinucleotides in a single genomic region can regulate gene transcription. Therefore, the complex relationship between DNA methylation and its high-precision genomic location limits the dynamic monitoring during tumor metastasis. There is currently a lack of excellent screening tools to achieve the regulation of methylation changes at specific genomic loci. Therefore, developing new screening tools to more deeply study the methylation characteristics of high-precision loci helps to accelerate the discovery of new genes silenced by hypermethylation during tumor metastasis, more comprehensively analyze the methylation pattern during tumor metastasis, and thus develop new treatment strategies. Summary of the Invention
[0004] Based on the above problems in the prior art, the present invention provides a composition for epigenetic editing, which includes a Cas functional sequence, a TET functional sequence, and an sgRNA targeting a target sequence site.
[0005] Wherein, the composition is a lentiviral vector, and the lentiviral vector further includes a U6 sequence and an MS2 stem loop; the Cas functional sequence is a dCas9 expression cassette, and the TET functional sequence is a TET1 active center expression cassette.
[0006] Wherein, the Cas functional sequence and the TET functional sequence are fused into a dCas9-tet1 sequence, the nucleotide sequence of the dCas9-tet1 sequence is as shown in SEQ ID NO.1, and the amino acid sequence of the dCas9-tet1 sequence is as shown in SEQ ID NO.2; the backbone nucleotide sequence of the sgRNA is as shown in SEQ ID NO.3.
[0007] The present invention also provides an epigenetic editing and cell selection method, which comprises the following steps:
[0008] Step S1: Infect a target cell line with lentivirus and screen out a labeled cell line that can stably express a labeled molecule and dCas9-tet1;
[0009] Step S2: Perform in vivo and in vitro proliferation cultures on the labeled cell line, and simultaneously perform target interference operations. After the proliferation culture, extract genomic DNA, perform methylation sequencing on the genomic DNA, and simultaneously perform RNA sequencing;
[0010] Step S3: Integrate the genomic DNA methylation sequencing results and RNA sequencing results of the target cell line without target interference operations, and the corresponding sequencing results of Step S2, perform correlation calculations based on the expression of relevant genes, and select target genes according to the calculation results;
[0011] Step S4: Select corresponding target sites according to the target genes selected in Step S3, design corresponding sgRNAs according to the target sites, and then add positive controls and sgRNAs without target sites to form a full amount of sgRNAs;
[0012] Step S5: Synthesize each sgRNA in the full amount of sgRNAs respectively and construct corresponding lentiviral vectors, then infect the target cell line with the corresponding lentiviral vectors, and after screening out the stably expressed cells, perform in vivo proliferation cultures respectively, and then select the best target sites according to the expression of the corresponding epigenetic characteristics of the target genes, that is, the library is constructed; the library contains the above-mentioned compositions corresponding to all target genes of the target cell line;
[0013] Step S6: Extract the compositions corresponding to the target genes or target sites from the library according to the target genes, perform demethylation editing on the target genes or target sites of the target cell line, and select cells with corresponding epigenetic characteristics according to the epigenetic characteristics corresponding to the target genes, and then downstream research can be carried out.
[0014] Wherein, the labeled molecule in Step S1 is a TROP2 molecule, and the CDS region sequence source of the TROP2 clone is CCDS609.1.
[0015] Wherein, the target interference operations in Step S2 include drug injection and drug addition treatments.
[0016] Wherein, in Step S4, one or more corresponding target sites are selected according to each target gene, and 6-8 corresponding sgRNAs are designed for each target site.
[0017] The beneficial effects of the present invention are as follows: The composition provided by the present invention can achieve the rewriting of the methylation degree at a specific genomic locus with high precision under the guidance of sgRNA, thereby regulating the expression level of the target gene. Through this composition and the method for selecting cells with the desired phenotype, high-throughput in vivo screening of animals can be achieved, and the methylation change characteristic map of high-precision genomic loci can be mapped, which helps to accelerate the discovery of new genes silenced by hypermethylation during tumor metastasis and more comprehensively analyze the methylation pattern during tumor metastasis. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 It is a schematic structural diagram of the dCas9-tet1 tool enzyme.
[0019] Figure 2 It is a fluorescence detection image of cells successfully transfected with sgRNA.
[0020] Figure 3 It is the expression of mRNA levels after demethylation regulation of CX3CL1 in four cell lines, MCF-7, MDA-MD-486, T47D, and HEK293, using different versions of gRNA.
[0021] Figure 4 It is a schematic diagram of the experimental process for mapping the genomic methylation map by mouse tumor sequencing after treatment with different concentrations of DAC.
[0022] Figure 5 It is a statistical chart of the number distribution of DMR anchor regions in the treatment group and the control group (including promoter, exon, intron, CGI, CGI shore, repeat, TSS, TES).
[0023] Figure 6 It is a clustering heat map of the DMR methylation levels in the treatment group and the control group.
[0024] Figure 7 It is a box plot of DMR distribution.
[0025] Figure 8 It is a Venn diagram of the target genes of the integrated sequencing results, TCGA, and METABRIC databases;
[0026] Figure 9 It is a GO enrichment bubble chart of the library candidate targets.
[0027] Figure 10 It is a KEGG pathway enrichment bubble chart.
[0028] Figure 11 It is a schematic diagram of the library screening process.
[0029] Figure 12It is the result graph of the phenotypic effect of the candidate target STAT5A on tumor cells. Overexpression of STAT5A promotes tumor cell proliferation.
[0030] Figure 13 It is the result graph of the phenotypic effect of the candidate target STAT5A on tumor cells. Overexpression of STAT5A promotes colony formation.
[0031] Figure 14 It is the result graph of inhibiting the migration and invasion abilities of tumor cells.
[0032] Figure 15 It is the result graph of STAT5A research. Activating STAT5A through VP64 or using its natural ligand prolactin (PRL) to activate STAT5A can increase phosphorylated STAT5A, and the enhancing effect of the ligand is time-dependent.
[0033] Figure 16 It is the result graph of STAT5A research. Immunofluorescence detection indicates that PRL can promote the nuclear entry of phosphorylated STAT5A to regulate the expression of downstream genes.
[0034] Figure 17 It is the immunoblot of the STAT5A expression result.
[0035] Figure 18 It is the immunofluorescence detection of the STAT5A expression result. It is found that phosphorylated STAT5A can increase the expression of E-cadherin.
[0036] Figure 19 It is the result graph of STAT5A research. Knockdown of STAT5A can block the increase in E-cadherin expression induced by PRL.
[0037] Figure 20 It is the result graph of monitoring tumor size by in vivo animal bioluminescence imaging technology. It is found that knockdown of STAT5A can inhibit orthotopic implanted breast tumors and distant metastases in mice.
[0038] Figure 21 It is the graph of fluorescence imaging for detecting the number of lung metastases.
[0039] Figure 22 It is the graph of the number of liver metastases.
[0040] Figure 23 It is the graph of the number of abdominal metastases.
[0041] Figure 24 It is the graph of immunohistochemical staining of in situ tumor tissues to evaluate the expression of two indicators, Ki67 and vimentin. Normal control n = 3, shRNA n = 5.
[0042] The colors in some of the attached drawings are part of the experimental results, and color drawings are used to clearly display the experimental results. Detailed implementation manners
[0043] The technical solutions of the present invention will be described below in conjunction with specific embodiments.
[0044] Embodiment 1: A composition for epigenetic editing.
[0045] As Figure 1 shown, a composition for epigenetic editing, the composition is a lentiviral vector, which includes a Cas functional sequence, a TET functional sequence, a U6 sequence, an MS2 stem loop and an sgRNA targeting a target sequence site; the Cas functional sequence is a dCas9 expression cassette, and the TET functional sequence is a TET1 active center expression cassette; wherein, the Cas functional sequence and the TET functional sequence are fused into a dCas9-tet1 sequence, the nucleotide sequence of the dCas9-tet1 sequence is shown in SEQ ID NO.1, and the amino acid sequence of the dCas9-tet1 sequence is shown in SEQ ID NO.2; the backbone nucleotide sequence of the sgRNA is shown in SEQ ID NO.3.
[0046] Embodiment 2: A method for epigenetic editing and cell selection.
[0047] This embodiment provides a method for epigenetic editing and cell selection using the composition described in Embodiment 1, which includes the following steps:
[0048] Step S1, infect the target cell line with lentivirus, and screen out the labeled cell line that can stably express the labeled molecule and dCas9-tet1; the labeled molecule is a TROP2 molecule, and the CDS region sequence source of the TROP2 clone is CCDS609.1;
[0049] Step S2, perform in vivo and in vitro proliferation cultures on the labeled cell line, and at the same time perform target interference operations, the target interference operations include drug injection and drug addition treatments. After proliferation culture, extract genomic DNA, and perform methylation sequencing on the genomic DNA, and at the same time perform RNA sequencing;
[0050] Step S3, integrate the genomic DNA methylation sequencing results and RNA sequencing results of the target cell line without target interference operations, and the corresponding sequencing results in Step S2, and perform correlation calculation according to the relevant gene expression conditions, and select target genes according to the calculation results;
[0051] Step S4: Based on the target genes selected in step S3, corresponding target sites are further selected. One or more corresponding target sites are selected for each target gene. Then, corresponding sgRNAs are designed according to the target sites. 6-8 corresponding sgRNAs are designed for each target site. Then, positive controls and sgRNAs without target sites are added to form the full amount of sgRNAs.
[0052] Step S5: Each sgRNA in the full amount of sgRNAs is synthesized separately and the corresponding lentiviral vector is constructed. Then, the target cell line is infected with the corresponding lentiviral vector. After the stably expressing cells are screened out, they are respectively cultured in vivo for proliferation. Then, the best target sites are selected according to the expression of the corresponding epigenetic characteristics of the target gene, that is, the library is constructed. The library contains the compositions as described in Example 1 corresponding to all target genes of the target cell line.
[0053] Step S6: According to the target gene, the composition corresponding to the target gene or the target site is extracted from the library, and demethylation editing is performed on the target gene or the target site of the target cell line. For example, different versions of gRNAs are used to perform demethylation regulation on the CX3CL1 gene in four cell lines, namely MCF-7, MDA-MD-486, T47D, and HEK293. The expression of its mRNA level is as Figure 3 shown. According to the epigenetic characteristics corresponding to the target gene, the target cell line is selected, and the cells with the corresponding epigenetic characteristics are selected, and then downstream research can be carried out. For example, it can be used for high-throughput screening, identifying one or more genes that enhance the desired phenotype of tumor cells. Exemplary phenotypes for screening or selection include the formation of an inhibitory tumor microenvironment, resistance to the effect of immunotherapy, promotion of pre-metastatic lung microenvironment changes, promotion of pre-metastatic liver microenvironment changes, increased tumor cell expansion / proliferation, and combinations thereof.
[0054] Cells with the desired phenotype are selected by injecting the cell population via the tail vein or the mammary fat pad and then combining with specific treatment means. Then, cells with the desired phenotype are selected by flow cytometry-based or affinity-based sorting, immunomarker-based selection, directed evolution, or a combination thereof.
[0055] In addition, the method can include identifying the sgRNA expression cassette present in the selected cells. This identification can be achieved by sequencing the genomic DNA of the selected cells (e.g., at or near the genomic integration region). Once the sgRNA expression cassette present in the selected cells is known, the gene that enhances the desired phenotype can be identified as the gene targeted by the guide RNA encoded by the sgRNA expression cassette. Abundance analysis is performed with the sgRNAs in the control group library, and negative screening can also be performed in the desired phenotype.
[0056] Example 3: The epigenetic editing and cell selection methods in Example 2 were implemented using triple-negative breast cancer cells (TNBC) as the target cell line.
[0057] This example includes the following steps:
[0058] Step S1: The murine TNBC cell line EO771 was infected with lentivirus, and a labeled cell line that could stably express human TROP2 molecule and dCas9-tet was screened out; the CDS region sequence source of the TROP2 clone was CCDS609.1. After stable screening, this cell line carried an mcherry fluorescent label, as Figure 2 shown, and this cell line was denoted as EO771-TDM.
[0059] Step S2: The EO771-TDM cell line was cultured in mice in vivo to construct a mouse mammary in-situ transplantation tumor. C57BL / 6 female mice aged 6-8 weeks were selected. At the same time, another EO771-TDM cell line was cultured in vitro for proliferation using a medium;
[0060] The mice were subcutaneously injected with DAC. The experimental group concentrations were set at 0.625 mg / kg and 1.25 mg / kg. The mouse tumors were collected for flow sorting and genomic DNA was extracted, as Figure 4 shown; the in vitro cultured EO771-TDM cell line was treated with DAC in vitro. The drug concentrations were set at 400 nM, 800 nM, and 1600 nM. After culturing for 7 days, genomic DNA was extracted. Both the subcutaneous injection of DAC and the in vitro DAC treatment were target interference operations. The extracted genomic DNA was subjected to restriction enzyme-bisulfite targeted sequencing (RRBS), and at the same time, RNA sequencing (RNA-seq) was performed;
[0061] Step S3: The methylation sequencing results and RNA-seq results of human TNBC without target interference operations were obtained from the TCGA and METABRIC databases. Correlation calculations were performed based on the expression of immune-related genes, and the sequencing results corresponding to Step S2 were integrated. According to the analysis results, 473 target genes were selected for library design;
[0062] Step S4: According to the target genes selected in Step S3, the corresponding target sites were further selected. One or more corresponding target sites were selected for each target gene, and a total of 670 target sites were selected. Then, the corresponding sgRNAs were designed according to the target sites. 6-8 corresponding sgRNAs were designed for each target site. In addition, positive controls and sgRNAs without target sites were added to form the full amount of sgRNAs. The total number of the full amount of sgRNAs was 5898;
[0063] Step S5: Synthesize each sgRNA in the full-length sgRNA library separately and construct the corresponding lentiviral vector. Then, infect the target cell line with the corresponding lentiviral vector. After screening for stably expressing cells with puromycin, sequence to identify the library coverage depth, ensuring that the library coverage is ≥200, to obtain the library EO771 cell line.
[0064] As Figures 5 - 11 shown, use the library EO771 cell line to construct a mouse orthotopic transplantation tumor model. After grouping the mice, perform treatment: the control group is injected with IgG4 antibody, and the experimental group is injected with αTROP2 CAR-T and αPD-1 antibody (Camrelizumab); during the treatment period, detect the general indicators and tumor growth of the mice; after 21 days, euthanize the mice, extract the orthotopic tumor tissue and lung metastatic tumor tissue for cell sorting, DNA extraction, and PCR amplification. Sequence the amplification products to analyze the changes in sgRNA, and construct a lentiviral high and low expression system for a single target for each of the changes in the corresponding sgRNA of the target gene and the expression of epigenetic characteristics; construct a mouse transplantation tumor model with the cell line intervened by a single target and repeat the above treatment plan to detect the changes in the anti-tumor effect; use flow cytometry to analyze the subtypes of tumor-infiltrating immune cells in the single-target model, and select the best target site based on the comprehensive anti-tumor effect, that is, complete the construction of the library; the library contains the compositions as described in Example 1 corresponding to all target genes of the target cell line.
[0065] Step S6: Extract the composition corresponding to the target gene or target site from the library according to the target gene, perform demethylation editing on the target gene or target site of the target cell line, and select the target cell line according to the epigenetic characteristics corresponding to the target gene to select cells with the corresponding epigenetic characteristics, and then downstream research can be carried out.
[0066] Example 4: Use the library constructed in Example 3 to perform epigenetic editing and cell selection of the STAT5A gene and conduct downstream research.
[0067] In this example, the STAT5A gene is used as the target gene, and it is clarified that STAT5A can reshape the immune microenvironment and improve the response to combined immunotherapy.
[0068] Extract the composition corresponding to the STAT5A gene from the library, construct a lentivirus for overexpressing or knocking down STAT5A, and use RT-qPCR and WB methods to detect whether the system construction is successful.
[0069] Use the above tumor cell lines with knocked-down or overexpressed STAT5A to form orthotopic tumors in the mammary glands of 6-8-week-old C57BL / 6 female mice; after tumor formation, administer Camrelizumab intraperitoneally at 10 mg / kg every 2 days for 14 days, and record the tumor volume and mouse body weight.
[0070] Flow cytometry was used to detect the number and phenotypic changes of tumor-infiltrating CAR-T cells, and the changes of various T cells, B cells, NK cells, macrophages, etc. were analyzed. The cells were divided into two groups for detection, and the detection indicators and cell colorings were as follows: single-label group: (1) Blank; (2) CD45-FITC; (3) CD11b-APC (4) CD3-APC; (5) CD4-PE; (6) F4 / 80-AlexFluro 700 (7) CD8-Alex Fluro 7. Group 1: live+CD45+CD3+CD8 CD4 NK1.1 CD19 (6 colors); Group 2: live+CD45+CD11b+F4 / 80+CD86 CD163 (5 colors).
[0071] Further explore the molecular mechanism of STAT5A:
[0072] Analysis of key regulatory targets and pathways of STAT5A: RNA-seq sequencing technology and Cut&tag sequencing technology were used to conduct downstream molecule exploration on the STAT5A high-expression or knockdown group, and KEGG, GO, and GSEA analysis methods were successively used to discover and identify key regulatory pathways. The key genes with statistical differences found through analysis will be verified by RT-qPCR and WB.
[0073] At the cell line level, clarify the effect of STAT5A on EMT, and detect the protein level expression of several core indicators such as vimentin and E-cadherin. The results are as Figures 12 - 24 shown.
[0074] The above embodiments provided by the present invention are only used to illustrate rather than limit the technical solutions of the present invention. Although the above embodiments have described the present invention in detail, those skilled in the relevant art should understand that: the present invention can be modified or equivalently replaced, but any modification and partial replacement that do not depart from the spirit and scope of the present invention should be covered within the scope of the claims of the present invention.
Claims
1. A composition for epigenetic editing, characterized in that, It includes a Cas functional sequence, a TET functional sequence, and an sgRNA targeting the target sequence site.
2. The composition for epigenetic editing according to claim 1, wherein The composition is a lentiviral vector, and the lentiviral vector further includes a U6 sequence and an MS2 stem loop; the Cas functional sequence is a dCas9 expression cassette, and the TET functional sequence is a TET1 active center expression cassette.
3. The composition for epigenetic editing according to claim 1, wherein The Cas functional sequence and the TET functional sequence are fused into a dCas9-tet1 sequence. The nucleotide sequence of the dCas9-tet1 sequence is shown in SEQ ID NO.1, and the amino acid sequence of the dCas9-tet1 sequence is shown in SEQ ID NO.2; the backbone nucleotide sequence of the sgRNA is shown in SEQ ID NO.
3.
4. An epigenetic editing and cell selection method, characterized in that, It includes the following steps: Step S1: Infect the target cell line with lentivirus and screen out the labeled cell line that can stably express the labeled molecule and dCas9-tet1. Step S2: Perform in vivo and in vitro proliferation cultures on the labeled cell line, and at the same time perform target interference operations. After the proliferation culture, extract genomic DNA, perform methylation sequencing on the genomic DNA, and perform RNA sequencing at the same time. Step S3: Integrate the genomic DNA methylation sequencing results and RNA sequencing results of the target cell line without target interference operations and the corresponding sequencing results in Step S2, perform correlation calculations based on the expression of relevant genes, and select target genes according to the calculation results. Step S4: Select the corresponding target sites according to the target genes selected in Step S3, design the corresponding sgRNAs according to the target sites, and then add positive controls and sgRNAs without target sites to form a full set of sgRNAs. Step S5: Synthesize each sgRNA in the full set of sgRNAs and construct the corresponding lentiviral vector, then infect the target cell line with the corresponding lentiviral vector, and after screening out the stably expressed cells, perform in vivo proliferation cultures respectively, and then select the best target sites according to the expression of the corresponding epigenetic characteristics of the target gene, that is, the library is constructed; the library contains the compositions as described in any one of claims 1-3 corresponding to all target genes of the target cell line. Step S6: Extract the composition corresponding to the target gene or the corresponding target site from the library according to the target gene, perform demethylation editing on the target gene or the target site of the target cell line, and select the cells with the corresponding epigenetic characteristics according to the epigenetic characteristics corresponding to the target gene, and then downstream research can be carried out.
5. An epigenetic editing and cell selection method according to claim 4, characterized in that, The labeled molecule in Step S1 is a TROP2 molecule, and the CDS region sequence source of the TROP2 clone is CCDS609.
1.
6. An epigenetic editing and cell selection method according to claim 4, characterized in that, The target interference operation in Step S2 includes drug injection and drug addition treatment.
7. An epigenetic editing and cell selection method according to claim 4, characterized in that In Step S4, one or more corresponding target sites are selected according to each target gene, and 6-8 corresponding sgRNAs are designed for each target site.