CRISPR library screening method of genes related to ovarian cancer cell growth, tumor formation and immune escape

Abstract

The invention belongs to the technical field of tumor treatment, and particularly relates to a CRISPR library screening method of genes related to ovarian cancer cell growth, tumor formation and immune escape. The sgRNA library comprises sgRNA which is selected from 80 genes in a MusCK library in a targeting manner. According to the invention, a targeted MusCK library of 922 genes related to the starting, progression and immunoregulation of tumors is used, and a P53- / -Carm1OE Ccne1OE KrasOE mouse ovarian cancer cell line, a C57BL / 6 mouse and a BALB / c-nu mouse are respectively adopted for high-throughput screening, so that the immunoregulation of tumors is realized. A series of confirmed and unverified key genes for high-grade serous ovarian cancer cell growth, tumor formation and immune escape function exertion are successfully screened, and an important theoretical and experimental foundation is laid for finding a novel treatment target of the high-grade serous ovarian cancer; and breakthrough in targeted therapy of high-grade serous ovarian cancer is facilitated.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of tumor treatment, and particularly relates to a CRISPR library screening method for genes related to ovarian cancer cell growth, tumor formation and immune escape. BACKGROUND

[0002] Ovarian cancer is a highly lethal gynecological malignancy and the fifth leading cause of cancer death in women. Among them, high-grade serous ovarian cancer (HGSOC) is the most common subtype, accounting for more than 70% of all ovarian cancer cases.

[0003] The current first-line standard treatment includes cytoreductive surgery and platinum-taxane maintenance chemotherapy. After first-line treatment, 60%~70% of patients will relapse under optimal cytoreduction (residual disease <1 cm) and 80%~85% of patients will relapse under suboptimal cytoreduction (residual disease >1 cm), resulting in a five-year survival rate of only about 45%. The development of first-line maintenance therapy attempts to extend this interval. Approved maintenance therapy regimens, including bevacizumab or PARP inhibitors, have been shown to prolong progression-free survival (PFS), but fail to significantly improve overall survival (OS), indicating the need for more effective maintenance therapy. Currently, most clinical trials focus on targeted treatment methods, including recent attempts to introduce immunotherapy into the field of ovarian cancer treatment.

[0004] Therefore, it is of great significance to seek high-grade serous ovarian cancer-specific treatment targets for the treatment of high-grade serous ovarian cancer. High-throughput CRISPR library screening technology is a new method for finding tumor targets, but because the current in vivo CRISPR library screening mainly uses immortalized tumor cell lines injected into nude mice for research, it lacks a tumor immune microenvironment; and the classic syngeneic mouse model of ovarian cancer (such as ID8) cannot well simulate the genotype and cell origin of HGSOC, so the current in vivo CRISPR library screening of high-grade serous ovarian cancer cannot meet the needs of screening high-grade serous ovarian cancer tumor immune-related targets. SUMMARY

[0005] In response to the need to develop high-grade serous ovarian cancer tumor immune-related targets using in vivo CRISPR library screening, the present application provides an sgRNA library, based on which a gene-edited high-grade serous ovarian cancer cell model is established, and a CRISPR library screening method for genes related to ovarian cancer cell growth, tumor formation and immune escape is carried out. To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows.

[0006] The first object of the present application is to provide an sgRNA library for screening immune-related targets of high-grade serous ovarian cancer tumor, which is a Focused MusCK plasmid library; the Focused MusCK plasmid library comprises sgRNAs targeting 80 genes selected from the MusCK library; and the MusCK library is Liu Lab MusCK Library A on Addgene.

[0007] The target sequence of the Focused MusCK plasmid library is selected from Table 3.

[0008] The sgRNA library provided by the present application is constructed by targeting key genes of high-grade serous ovarian cancer cell growth, tumor formation and immune escape, and comprises 80 gene targets closely related to tumor microenvironment interaction, and the 80 gene targets have been verified in library screening. Each gene is equipped with 3 high-efficiency sgRNA sequences verified by off-target effect, ensuring the coverage and reliability of the screening process. Therefore, the sgRNA library provided by the present application can respond to the need for developing high-grade serous ovarian cancer tumor immune-related targets by using in vivo CRISPR library screening.

[0009] The second object of the present application is to provide the application of the sgRNA library in screening genes related to ovarian cancer cell growth, tumor formation and tumor immune escape.

[0010] The third object of the present application is to provide the application of the sgRNA library in preparing a kit for screening immune-related genes of high-grade serous ovarian cancer with CCNE1 overexpression.

[0011] The fourth object of the present application is to provide a CRISPR library screening method of candidate genes related to ovarian cancer cell growth, tumor formation and tumor immune escape, comprising the following steps: Using the MusCK library, perform pan-cancer development CRISPR library screening of high-grade serous ovarian cancer cells, C57BL / 6 mice and BALB / c-nu mice based on gene editing.

[0012] Using bioinformatics analysis, identify candidate genes related to high-grade serous ovarian cancer cell growth, tumor formation and tumor immune escape from the results of the pan-cancer development CRISPR library screening.

[0013] According to the results of the pan-cancer development CRISPR library screening, perform high-grade serous ovarian cancer focused screening to obtain the sgRNA library.

[0014] Based on the sgRNA library, a high-grade serous ovarian cancer focused validation screening based on the gene editing of the mouse high-grade serous ovarian cancer cells, C57BL / 6 mice and BALB / c-nu mice is carried out.

[0015] The results of the high-grade serous ovarian cancer focused validation screening are analyzed by bioinformatics to identify candidate genes related to the growth, tumor formation and tumor immune escape of high-grade serous ovarian cancer cells.

[0016] Preferably, the gene-edited mouse high-grade serous ovarian cancer cells are P53 - / - Carm1 OE Ccne1 OE Kras OE Mouse ovarian cancer cells.

[0017] The P53 - / - Carm1 OE Ccne1 OE Kras OE Mouse ovarian cancer cells are constructed by the following method, comprising the following steps: An ex vivo mouse primary oviduct epithelial cell culture organoid is obtained.

[0018] After knocking out the P53 gene of the ex vivo mouse primary oviduct epithelial cell culture organoid, a 2D culture method is used to construct P53 - / - Mouse oviduct epithelial cell lines.

[0019] Overexpression of Carm1, Ccne1 and Kras genes is carried out on the P53 - / - Mouse oviduct epithelial cell lines by lentivirus transfection.

[0020] P53 - / - Carm1 OE Ccne1 OE Kras OE Mouse ovarian cancer cells are obtained by flow sorting and WB validation screening.

[0021] The P53 - / - Carm1 OE Ccne1 OE Kras OEThe mouse ovarian cancer cell line is derived from C57 mouse oviduct epithelium, and is genetically edited with high-grade serous ovarian cancer common gene mutations, P53 deletion, Carm overexpression, Ccne1 overexpression, Kras overexpression. Compared with traditional ovarian cancer cell lines (such as ID8), it better simulates the genetic mutations and cell origin of high-grade serous ovarian cancer. The cell line supports C57 homologous mouse transplantation, and retains the complete immune microenvironment of mice compared with the heterotransplantation of nude mice using human ovarian cancer cell lines.

[0022] Preferably, the P53 - / - Carm1 OE Ccne1 OE Kras OE The mouse ovarian cancer cell is used to construct a high-grade serous ovarian cancer cell model. The construction of the genetically engineered ovarian cancer cell model follows the core driving mechanism of high-grade serous ovarian cancer, and adopts a four-dimensional carcinogenic module combined regulation strategy:

[0023] (1) Genomic stability disruption: P53 gene knockout mediated by CRISPR / Cas9 simulates the characteristics of HGSOC high-frequency TP53 mutation (>96%).

[0024] (2) Epigenetic reprogramming: overexpression of Carm1 (coactivator-associated arginine methyltransferase 1) induces abnormal methylation of histone H3R17 site, activates Wnt / β-catenin pathway and promotes stem cell characteristics.

[0025] (3) Cell cycle deregulation: Ccne1 gene overexpression (>5 copies) leads to G1 / S checkpoint defects and establishes a chromosomal instability phenotype.

[0026] (4) Microenvironment remodeling: Kras overexpression conditionally activates the IL-6 / STAT3 paracrine signal to promote tumor-associated fibroblast activation.

[0027] Preferably, the candidate genes include ovarian cancer in vivo growth targets and ovarian cancer immune targets.

[0028] The ovarian cancer in vivo growth targets include at least one of HMBS, ARHGEF16, CLDN3, KMT2C, CDKN1B, CCDC6, QK, JADE1, NFE2L3 and SETD1; and the ovarian cancer immune targets include at least one of BATF, KDM2B, TARS2, PSMB10, NTRK1, ADAMTS12, HS6ST2, POLB, NID2 and FAM178B.

[0029] Compared with the prior art, the application has the following beneficial effects: 1. The application provides an sgRNA library for screening high-grade serous ovarian cancer tumor immune-related target points, the sgRNA library comprising sgRNAs targeting 80 genes selected from a MusCK library; the MusCK library is Liu Lab MusCK Library A on Addgene. The target sequence of the Focused MusCK plasmid library is selected from Table 3. The sgRNA library established by the application is composed of candidate genes screened out by the MusCK library in the first round of in vivo CRISPR screening, the genes targeted by the sgRNA library are more strongly related to the correlation between HGSOC tumor growth and tumor immune function, thereby the range of screened genes can be reduced, and the accuracy and reliability of target point screening can be improved. The gRNA library and the construction method thereof provided by the application meet the simulation of the genotype and cell origin of HGSOC and the complete HGSOC tumor immune microenvironment at the same time, and thus the need for screening high-grade serous ovarian cancer tumor immune-related target points can be met.

[0030] 2. The application provides a CRISPR library screening method for ovarian cancer cell growth, tumor formation and immune escape related genes. The application expands the application of the MusCK library in screening high-grade serous ovarian cancer (HGSOC) cell growth, tumor formation and immune escape function, establishes a Focused MusCK library using screened candidate genes, performs a second round of screening, and obtains a series of key genes that have been verified and not verified, thereby laying an important theoretical and experimental foundation for discovering new high-grade serous ovarian cancer treatment targets, and facilitating breakthroughs in high-grade serous ovarian cancer targeted therapy.

[0031] 3. The application also provides a P53 - / - Carm1 OE Ccne1 OE Kras OE mouse ovarian cancer cell line, the P53 - / - Carm1 OE Ccne1 OE Kras OE mouse ovarian cancer cell line better simulates the common gene mutations and cell origins of HGSOC. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 A flowchart of the CRISPR library screening based on in-vitro growth screening in the application.

[0033] Figure 2 A box plot of the sgRNA frequency distribution of the MusCK library of samples at different time points in the application.

[0034] Figure 3 Volcano plot of differentially expressed genes between samples at different time points in the present application.

[0035] Figure 4 RRA score ranking of corresponding genes generated from the enrichment of sgRNAs based on in vitro growth screening in the present application, wherein, Figure 4 Figure A in the present application is the top ten missing genes ranked by RRA score; Figure 4 Figure B in the present application is the top ten enriched genes ranked by RRA score.

[0036] Figure 5 Relative abundance of sgRNAs of the top 5 genes ranked by RRA score in the present application.

[0037] Figure 6 KEGG pathway enrichment analysis of the top 100 genes ranked by the most significant deletion under in vitro growth screening in the present application.

[0038] Figure 7 Venn diagram of missing genes (FDR < 5%) in the first round of screening and the second round of repeated screening of the CRISPR library based on in vitro growth screening in the present application.

[0039] Figure 8 Flow chart of CRISPR library screening based on in vivo growth and immune stress screening in the present application.

[0040] Figure 9 P53 introduced into MusCK library in the present application - / - Carm1 OE Ccne1 OE Kras OE Statistical analysis of the diameters of mouse orthotopic transplanted tumors of ovarian cancer cell lines, wherein, Figure 9 Figure A in the present application is the orthotopic transplanted tumors of nude mice, Figure 9 Figure A in the present application is the orthotopic transplanted tumors of C57 mice, wherein the upper row is one repeat for each tumor, and there are 19 repeats, and the lower row is one repeat for each tumor, and there are 19 repeats; Figure 9 Figure B in the present application is a statistical analysis of the diameters of mouse orthotopic transplanted tumors.

[0041] Figure 10 Box plot of sgRNAs of MusCK library in different samples in the present application.

[0042] Figure 11 RRA score ranking of corresponding genes generated from the enrichment of sgRNAs based on immune stress screening in the present application.

[0043] Figure 12KEGG pathway enrichment analysis of the top 50 genes with the most significant deletions in the immune pressure screen in the present application.

[0044] Figure 13 P53 - / - Carm1 OE Ccne1 OE Kras OE KDM2B - / - Mouse ovarian cancer organoid system, verification of the function of KDM2B in tumor immune escape; Figure 13 A figure in the present application is P53 - / - Carm1 OE Ccne1 OE Kras OE and P53 - / - Carm1 OE Ccne1 OE Kras OE KDM2B - / - Mouse ovarian cancer organoid bright field figure, wherein the left and right two figures are P53 - / - Carm1 OE Ccne1 OE Kras OE KDM2B - / - Mouse ovarian cancer organoid 1# monoclonal stable strain and 2# monoclonal stable strain; Figure 13 B figure in the present application is P53 - / - Carm1 OE Ccne1 OE Kras OE KDM2B - / - Sanger sequencing verification of the genotype of the mouse ovarian cancer organoid; Figure 13 C figure in the present application is P53 - / - Carm1 OE Ccne1 OE Kras OE and P53 - / - Carm1 OE Ccne1 OE Kras OE KDM2B - / - Mouse ovarian cancer organoid tumor figure after subcutaneous injection of C57BL / 6 mice and BALB / c-nu mice for 16 days, respectively, wherein the first row is the control group, each tumor is one repeat, C57BL / 6 mice and BALB / c-nu mice each group has 4 repeats, the second and third rows are KDM2B knockout groups, each tumor is one repeat, C57BL / 6 mice have a total of 6 repeats, and BALB / c-nu mice have a total of 8 repeats; Figure 13 D figure in the present application isFigure 13 Measurement of tumor diameter and statistical graph in C figure in FIG. 1. DETAILED DESCRIPTION

[0045] The application will be described in detail below with reference to the drawings and specific examples, but should not be understood as limiting the application. If not specifically stated, the technical means used in the following examples are conventional means known to those skilled in the art, and the materials, reagents, etc. used in the following examples, if not specifically stated, can be obtained from commercial channels.

[0046] Example 1: Construction of P53 - / - Carm1 OE Ccnel OE Kras OE Mouse ovarian cancer cell line 1, Extraction of P53 flox / flox Mouse oviduct primary cell culture organoid: Under the microscope, separate the mouse oviduct, and cut the distal oviduct into a 3.5 cm culture dish, add 200 μL collagenase / dispase on ice, cut the tissue, transfer into a 15 mL centrifuge tube, add 1 mL collagenase / dispase, digest at 37°C until there are no obvious tissue fragments. Terminate digestion with DPBS, centrifuge at 600 x g for 5 minutes, discard the supernatant. Add 500 μL red blood cell lysis solution and lyse on ice for 5 min, terminate with DPBS, centrifuge at 600 g for 5 minutes, discard the supernatant. Resuspend the pellet with medium and filter it through a 100 μm cell strainer, centrifuge at 600 x g for 5 minutes, discard the supernatant, resuspend the cell pellet with ovarian cancer organoid medium and matrigel at a volume ratio of 1:1, seed in a 24-well plate at a volume of 50 μL / well, and place in a 37°C incubator. After 15 minutes, add ovarian cancer organoid medium to obtain primary mouse oviduct organoids. The primary mouse oviduct organoids are ex vivo mouse primary oviduct epithelial cell culture organoids.

[0047] P53 flox / flox The mouse is from Nanmo Biological NM-CKO-18005.

[0048] The collagenase / dispase is from sigma 10269638001.

[0049] Ovarian cancer organoid culture medium: Advanced DMEM / F12 (Gibco 12634010) with 1x Glutamax (Gibco 35050061), 10 mM HEPES (Gibco 15630106), 100 ng / mL Wnt3a (Peprotech 315-20), 100 ng / mL Noggin (Peprotech 120-10c), 200 ng / mL Rspo1 (Peprotech 120-38), 1X B27 (Life Technologies 12587010), 1.25 mM N-Acetylcysteine (Sigma A9165-5G), 0.1 mg / mL Primocin (Invivogen ant-pm-1), 10 nM Nicotinamide (Sigma N0636), 500 nM A83-01 (wako 039-24111), 10 mM Y27623 (Wako 030-24021), 10 ng / mL FGF10 (Peprotech 100-26), and 50 ng / mL EGF (Peprotech 100-39) with water as solvent.

[0050] 2. Lentivirus packaging: 293T cells were recovered and subcultured to grow well and reach 80% confluence before lentivirus transfection. The specific method is as follows:

[0051] The lentivirus three-plasmid system was transfected according to the JetPRIME transfection reagent instructions. The total amount of lentivirus plasmid transfected was 20 pg, and the mass ratio of the three lentivirus plasmids was Cre-GFP: psPAX2: PMD2.G = 4:3:1. Fresh complete medium was replaced before transfection, and the medium was changed 6 h after transfection. Lentivirus supernatant was collected 72 h after transfection, centrifuged at 500xg for 10 min at 4°C to remove cell debris, and the supernatant was aliquoted and stored at -80°C. Cre virus liquid was obtained.

[0052] The 293T cells were from procell CL-0005.

[0053] The three lentivirus plasmids were Cre-GFP (Addgene #219536), psPAX2 (Addgene #12260), and PMD2.G (Addgene #12259).

[0054] 3. Cre lentivirus transfection of mouse oviduct epithelial organoids: Digest the organoids into single cells, resuspend the cells with Cre virus solution. Transfer the cell suspension to low attachment 24-well plate, centrifuge at 600g for 60min at 37C, transfer the 24-well plate to 37C incubator. 2h later, collect the cells into 15mL centrifuge tube, wash away the virus with 10mL DPBS, centrifuge at 600xg for 5min. Discard the supernatant, wash the cells with Ad+++ solution, centrifuge at 600xg for 5min. Discard the supernatant, resuspend the cell pellet with culture medium and Matrigel at a volume ratio of 1:1, seed the cells into 24-well plate at a volume of 50μL / well, put the plate into 37C incubator, 15min later, add the ovarian cancer organoid culture medium. 3 days later, pick the GFP positive organoids under microscope, culture them as 2D cells, construct them into P53 - / - Mouse oviduct epithelial cell line.

[0055] 4, Carm1 OE , Ccnel OE , Kras OE Lentivirus transfection of P53 - / - Mouse oviduct epithelial cell line: Confirm P53 - / - Mouse oviduct epithelial cells are in good condition, then they are inoculated in 10cm culture dishes; when the cell growth density reaches 50%, infect Carm1 OE , Ccnel OE , Kras OE Lentivirus, the final concentration of Polybrene is 10μg / mL, mix well to infect P53 - / - Mouse oviduct epithelial cells; 48h after infection, add Blasticidin S and G418 respectively to screen positive cells.

[0056] Flow sorting obtains single cells, 25 strains of single clone cells with normal cell morphology and rapid proliferation are selected for Western Blot experiment to detect the expression of Carm1, Ccnel and Kras, and the single clone with overexpression of Carm1, Ccnel and Kras is selected, and P53 - / - Carm1 OE Ccnel OE Kras OE Mouse ovarian cancer cell line.

[0057] Example 2: Expansion of MusCK library MusCK library is Liu Lab MusCK Library A on Addgene.

[0058] Electroporation of MusCK library: To thawed 25 μΐ of Lucigen Endura electrocompetent cells, add 2 μΐ of 50 ng / μΐ MusCK library DNA, mix and transfer to pre-chilled 1.0 mm electroporation cuvette, keep on ice. Place the cuvette in the Longza electroporator, adjust the voltage to 1.8 kV, time constant to 4 ms, capacitance to 25 μΐ, resistance to 200 Ω, and perform the electroporation. Within 10 seconds after the electric shock, add 975 μΐ of SOC medium (sigma S1797) to the cuvette; transfer the cells to a shaker tube, add 1 mL of SOC medium; place the shaker tube in a 37 °C, 250 rpm shaker for 1 hour. Mix all 8 mL of recovered cells; transfer 10 μΐ of mixed cells to 990 μΐ of SOC medium for 800-fold dilution, mix, and spread 20 μΐ of the diluted solution on a pre-warmed LB plate (10 cm) containing 100 mg / mL kanamycin. This results in a 40,000-fold dilution of the transformation, which is used to calculate the transformation efficiency. Distribute the remaining cells to 20 pre-warmed 15 cm LB plates at 37 °C, 400 μΐ of cell solution per plate. Incubate the cell-spreading plates at 30 °C for 14 hours. Count the colonies on the 40,000-fold dilution plate, multiply the colony number by 40,000 to get the total colony number; confirm that the total colony number can cover at least 200x sgRNAs of the library to ensure that the library is fully represented. To one 15 cm plate, add 7 mL of LB medium containing kanamycin (100 μg / L), scrape the colonies, and transfer the scraped cells to a sterile 1 L conical flask or bottle, use an additional 5 mL of LB medium to rinse the scraper and transfer to the bottle to collect all 20 plates of colonies; mix all scraped cells, use a magnetic stirrer to stir at room temperature for 1 hour to help break the cell clumps; transfer the cells to a pre-weighed centrifuge bottle, centrifuge at 7,000 x g, discard the supernatant, and collect the cell pellet.

[0059] Purify plasmid DNA using the large plasmid purification kit. PCR amplify the fragment containing sgRNA sequence information, the primers used in the first round of amplification are Primer_R and Primer_F.

[0060] The nucleotide sequence of Primer_R is shown in SEQ ID NO. 1: 5’-TCTACTATTCTTTCCCCTGCACTGTACCTGTGGGCGATGTGCGCTCTG-3’.

[0061] The nucleotide sequence of Primer_F is shown in SEQ ID NO. 2: 5'-AATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCG-3'.

[0062] The primers used in the second round of amplification were Cri_library_F, Cri_library_indexl and Cri_library_index2.

[0063] The nucleotide sequence of Cri_library_F is shown in SEQ D NO. 3: 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTCTTGTGGAAAGGACGAAACACCG-3'.

[0064] The nucleotide sequence of Cri_library_indexl is shown in SEQ D NO. 4: 5'-CAAGCAGAAGACGGCATACGAGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTATCACGTCTACTATTCTTTCCCCTGCACTGTACC-3'.

[0065] The nucleotide sequence of Cri_library_index2 is shown in SEQ D NO. 5: 5'-CAAGCAGAAGACGGCATACGAGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCGATGTTCTACTATTCTTTCCCCTGCACTGTACC-3'.

[0066] The amplification used Q5®High-Fidelity DNA Polymerase (Biolabs®), the amplification system is shown in Table 1, and the amplification procedure is shown in Table 2: Table 1 Amplification system Table 2 Amplification procedure 1% agarose gel electrophoresis detected high-throughput sequencing band size 200bp correct, sequencer for Illumina, high-throughput double-end sequencing, each end 150bp, high-throughput sequencing data first de-adapter and filter low-quality reads processing, then use FastQC to assess data quality, sequencing data Q30 and Error rate meet the requirements, can be used for subsequent data analysis. Use MAGeCK to compare the original sgRNA plasmid library sequencing results with the amplified sgRNA library plasmid, the reads number of the library plasmid shows normal distribution, good uniformity, coverage 100%. It is judged that the coverage of the sgRNA library plasmid is good, the reads are evenly distributed, and can be used for packaging sgRNA library lentivirus.

[0067] Wherein, Addgene: http: / / www.addgene.org / .

[0068] Liu Lab MusCK Library A: (Pooled Libraries #174196.

[0069] Endura cell source is Sigma LUC60242-1.

[0070] The super large plasmid purification kit is QIAGEN Plasmid Mega Kit, and the source is QIAGEN 12181.

[0071] Example 3: First round of in vitro growth-based CRISPR screening using MusCK library First, use 293T cells and packaging plasmids psPAX2, PMD2.G to package lentivirus, see example 1 for specific steps. In P53 - / - Carm1 OE Ccne1 OE Kras OE The MusCK library virus was introduced into mouse ovarian cancer cells by lentivirus at a low MOI (MOI=0.5), and the positive mixed library baseline cells were obtained by using 2ug / mL puromycin drug screening for 48 hours. The positive mixed clone cells were divided into 2 parts, each part was set as 3 groups: 1 part of cells as the baseline cells on day 0, immediately extract genomic DNA; 1 part of cells continue to culture and passage for 8 cell cycles (16 days), extract genomic DNA (as Figure 1 shown).

[0072] Example 4: In vitro screening of high-throughput sequencing data biological information analysis The cellular genomic DNA from Example 3 was amplified using PCR to create fragments containing sgRNA sequence information. Adapter and tag sequences were then ligated using PCR. The sequencing libraries prepared using the two-round amplification method were subjected to high-throughput sequencing, with a sequencing coverage depth of 1000× for each sgRNA. The raw FASTQ files generated by the Illumina platform were analyzed for sgRNA enrichment using MAGeCK software. The Count command was used for preliminary analysis of the high-throughput sequencing data, including the normalized read distribution of the samples. Figure 2 The results showed that, compared with baseline cells, cells cultured in vitro for 16 days exhibited both enrichment and loss of gRNA reads, with good reproducibility between groups. Differentially expressed genes were ranked using the `test` command to generate RRA scores. Based on the RRA calculation results, positive and negative selection (pos|score) were generated using the `test` command. Using P < 0.05 and lgFc > 2 as valid criteria for enrichment screening, the top ten enriched genes according to the RRA score were HMBS, ARHGEF16, CLDN3, KMT2C, CDKN1B, CCDC6, QK, JADE1, NFE2L3, and SETD1. Figure 4 (See Figure B in the original text). Using P < 0.05 and lgFc < -2 as valid criteria for negative selection, the top ten enriched genes according to the RRA Score were WARS, TUBA1C, RAN, CDT1, KIFC1, PRMT1, HAUS5, PCNA, SF3A2, and CCT2. Figure 4 Figure A in the diagram. Volcano plots were created using MAGeCKFlute to visualize differentially expressed genes. Figure 3 And view the gRNA rank of the top 4 genes in the positive and negative screening ( Figure 5 The results showed that the enrichment and deletion trends of the five gRNAs targeting each gene were consistent. KEGG pathway enrichment analysis was performed ( Figure 6 The results showed that the most significantly enriched pathways were all associated with fundamental functions that significantly affect cell survival and proliferation. Venn diagrams were plotted on the negative genes (LFC < 2 and P < 0.05) obtained from two repeated screenings. Figure 7 The results showed 444 overlapping genes, and the in vitro screening system exhibited good reproducibility.

[0073] Example 5: First-round CRISPR screening based on in vivo growth and immune stress using the Muskk library First, lentivirus was packaged using 293T cells and packaging plasmids psPAX2 and PMD2.G, following the same lentivirus packaging method as described in Example 2. (P53) - / - Carm1 OE Ccne1 OEKras OE The positive mixed library baseline cells were obtained by introducing MusCK library virus into mouse ovarian cancer cells with low MOI (MOI=0.5) by lentivirus and screening with puromicin for 5 days. The positive mixed clone cells were divided into 4 parts, each part into 3 groups: 1 part of cells as baseline cells on day 0, immediately extracting genomic DNA; 1 part of cells inoculated into immunocompetent C57BL / 6 mice; 1 part of cells inoculated into immunodeficient BALB / c-nu mice (Fig. 2). Figure 8 After 16 days of screening in the in vivo environment of mice, the transplanted tumors were removed by dissection. It was found that the diameter of the transplanted tumor in BALB / c-nu mice was larger than that in C57BL / 6 mice, suggesting that the complete immune system had an inhibitory screening effect on tumor cells. Figure 9

[0074] Example 6: In vivo growth and immune pressure screening of high-throughput sequencing data biological information analysis The genomic DNA of the cells and transplanted tumors in Example 5 was amplified by PCR to obtain fragments containing sgRNA sequence information, and the amplification primers were forward primer and reverse primer: The nucleotide sequence of the forward primer is shown in SEQ D NO. 6: 5'-GAGGGCCTATTTCCCATGATTC-3'.

[0075] The nucleotide sequence of the reverse primer is shown in SEQ D NO. 7: 5'-GTTGCGAAAAAGAACGTTCACGG-3'.

[0076] The adapter and tag sequences were connected by PCR, and the amplification primers were as follows: The nucleotide sequence of D501-F is shown in SEQ D NO. 8: 5'-AATGATACGGCGACCACCGAGATCTACACTATAGCCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTTGTGGAAAGGACGAAACACCG-3'.

[0077] The nucleotide sequence of D502-F is shown in SEQ D NO. 9: 5'-AATGATACGGCGACCACCGAGATCTACACATAGAGGCACACTCTTTCCCTACACGACGCTCTTCCGATCTTTGTGGAAAGGACGAAACACCG-3'.​

[0078] The nucleotide sequence of 5'-D503-F is shown in SEQ ID NO. 10: 5'-AATGATACGGCGACCACCGAGATCTACACCCTATCCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTTGTGGAAAGGACGAAACACCG-3'.

[0079] The nucleotide sequence of D504-F is shown in SEQ ID NO. 11: 5'-AATGATACGGCGACCACCGAGATCTACACGGCTCTGAACACTCTTTCCCTACACGACGCTCTTCCGATCTTTGTGGAAAGGACGAAACACCG-3'.

[0080] The nucleotide sequence of D701-R is shown in SEQ ID NO. 12: 5'-CAAGCAGAAGACGGCATACGAGATCGAGTAATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTACTTGCTATTTCTAGCTCTAAAAC-3'.

[0081] The nucleotide sequence of D702-R is shown in SEQ ID NO. 13: 5'-CAAGCAGAAGACGGCATACGAGATTCTCCGGAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTACTTGCTATTTCTAGCTCTAAAAC-3'.

[0082] The nucleotide sequence of D704-R is shown in SEQ ID NO. 14: 5'-CAAGCAGAAGACGGCATACGAGATGGAATCTCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTACTTGCTATTTCTAGCTCTAAAAC-3'.

[0083] The nucleotide sequence of D705-R is shown in SEQ ID NO. 15: 5'-CAAGCAGAAGACGGCATACGAGATTTCTGAATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTACTTGCTATTTCTAGCTCTAAAAC-3'.

[0084] The nucleotide sequence of D706-R is shown as SEQ ID NO. 16: 5'-CAAGCAGAAGACGGCATACGAGATACGAATTCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTACTTGCTATTTCTAGCTCTAAAAC-3'.

[0085] The nucleotide sequence of D707-R is shown as SEQ ID NO. 17: 5'-CAAGCAGAAGACGGCATACGAGATAGCTTCAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTACTTGCTATTTCTAGCTCTAAAAC-3'.

[0086] Both PCR amplification systems refer to PrimeSTAR® GXL DNA Polymerase (Takara R050A). The sequencing library is prepared for high-throughput sequencing, and the sequencing coverage depth of each sgRNA is 1000x. The original FASTQ file generated by the illumina platform is analyzed for sgRNA enrichment in different samples using the MAGeCK software. The Count command performs preliminary analysis on high-throughput sequencing data, and the standardized read distribution of the sample ( Figure 10 ) shows that the gRNA reads of the mouse transplanted tumor are missing as a whole compared with the baseline cells, and the C57BL / 6 mouse transplanted tumor with a perfect immune environment is more significant, suggesting that the complete immune system has a suppressive screening effect on tumor cells. The test command is used to generate a difference gene RRA Score for ranking. According to the RRA calculation result, the test command is used to generate a positive screening (pos|score) and a negative screening (neg|score). The negative screening is enriched in the top ten genes according to the RRA Score, which are BATF, KDM2B, TARS2, PSMB10, NTRK1, ADAMTS12, HS6ST2, POLB, NID2, and FAM178B ( Figure 11 ), which may be genes related to the function of the tumor against the immune environment in vivo. KEGG pathway enrichment analysis ( Figure 12 ) shows that the most significant pathway enriched is the tumor PD-1 / PD-L1 immune checkpoint pathway, which plays an important role in tumor immune escape.

[0087] Example 7: Establishment of a Focused MusCK library targeting ovarian cancer cell growth and immune genes According to the results of the two in vitro screening of MusCK library, the top 10 enriched pos. Ctrl of RRA Score were selected as positive controls, and the top 11 positive screening genes of LFC and the top 39 negative screening genes of LFC were selected according to the results of the two in vitro screening of MusCK library. According to the results of in vivo screening, the top 10 positive screening genes and the top 10 negative screening genes of immune screening were selected, and the Focused MusCK library targeting ovarian cancer cell growth and immune genes (see Table 3) was screened. The mixed library of custom 20bp sgRNA sequence DNA oligonucleotide was inserted into the lentiCRISPR-v2 vector by Gibson connection, and the Endura cells were used for library electroporation amplification.

[0088] Table 3 Focused MusCK library Note: Focused MusCK library refers to sgRNA library.

[0089] Among them, the lentiCRISPR-v2 vector is from Addgene #52961.

[0090] Example 8: Establish KDM2B knockout P53 - / - Carm1 OE Ccne1 OE Kras OE KDM2B - / - Mouse ovarian cancer organoid system, verify the function of KDM2B in tumor immune escape According to the results of immune screening, two KDM2B-targeted gRNA synthesis plasmids with the most significant deletion were selected, and 293T cells and packaging plasmids psPAX2 and PMD2.G were used for lentivirus packaging. After collecting the virus, P53 - / - Carm1 OE Ccne1 OE Kras OE Mouse ovarian cancer organoids, 72 hours after 10ug / mL puromycin drug screening for 48 hours, and picking single clone, Westerblot verification P53 - / - Carm1 OE Ccne1 OE Kras OE KDM2B - / - Mouse ovarian cancer organoid system (A figure in Figure 13 B figure in Figure 13 P53 - / - Carm1 OE Ccne1OE Kras OE and P53 - / - Carm1 OE Ccne1 OE Kras OE KDM2B - / - Mouse ovarian cancer organoids were administered subcutaneously to C57BL / 6 mice and BALB / c-nu mice, with a single injection dose of 1×10⁻⁶ cells. 6 Tumors were collected in 16 days. Figure 13 (See Figure C in the original text). Knockout of KDM2B resulted in almost complete absorption of the xenografts in C57BL / 6 mice; in contrast, KDM2B knockout did not affect the continued growth of xenografts in BALB / c-nu mice. This confirms the screening results indicating that KDM2B may play a crucial role in the tumor-versus-immune environment.

[0091] The experimental results above suggest that KDM2B may be a key gene in the tumor's immune environment.

[0092] It should be noted that when numerical ranges are involved in this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, this invention describes preferred embodiments.

[0093] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments, all of which fall within the scope of the invention.

Claims

1. A library of sgRNAs for screening immune-related targets in high-grade serous ovarian cancer tumors, characterized in that, the sgRNA library comprises sgRNAs targeting 80 genes selected from the MusCK library; the MusCK library is LiuLab MusCK Library A on Addgene; the target sequences of the sgRNA library are selected from the following table:

2. Use of the sgRNA library of claim 1 in screening genes related to ovarian cancer cell growth, tumor formation and tumor immune escape.

3. Use of the sgRNA library of claim 1 in preparing a kit for screening immune-related genes of high-grade serous ovarian cancer targeting CCNE1 overexpression.

4. A method of CRISPR library screening for candidate genes associated with ovarian cancer cell growth, tumor formation and tumor immune evasion, characterized by, comprising the following steps: using the MusCK library of claim 1, performing a pan-cancer development CRISPR library screening based on gene editing of mouse high-grade serous ovarian cancer cells, C57BL / 6 mice and BALB / c-nu mice; using bioinformatics to analyze the results of the pan-cancer development CRISPR library screening, identifying candidate genes related to high-grade serous ovarian cancer cell growth, tumor formation and tumor immune escape; based on the results of the pan-cancer development CRISPR library screening, performing a high-grade serous ovarian cancer focused screening, and preparing the sgRNA library; based on the sgRNA library, performing a high-grade serous ovarian cancer focused verification screening based on the gene editing of mouse high-grade serous ovarian cancer cells, C57BL / 6 mice and BALB / c-nu mice; using bioinformatics to analyze the results of the high-grade serous ovarian cancer focused verification screening, identifying candidate genes related to high-grade serous ovarian cancer cell growth, tumor formation and tumor immune escape.

5. The method of claim 4, wherein, The genetically edited mouse high-grade serous ovarian cancer cells are P53 - / - Carm1 OE Ccne1 OE Kras OE Mouse ovarian cancer cells; The P53 - / - Carm1 OE Ccne1 OE Kras OE The method for constructing mouse ovarian cancer cells comprises the following steps: obtaining an ex vivo mouse primary oviduct epithelial cell culture organoid; After knocking out P53 gene in the ex vivo mouse primary oviduct epithelial cell culture organoids, 2D culture method was used to construct P53 - / - Mouse oviduct epithelial cell line; In the P53 - / - Mouse oviduct epithelial cell lines were transfected with lentivirus overexpressing Carm1, Ccnel and Kras genes; P53 - / - Carm1 OE Ccne1 OE Kras OE Mouse ovarian cancer cells.

6. The method of claim 5, wherein, The P53 - / - Carm1 OE Ccne1 OE Kras OE Mouse ovarian cancer cells were used to construct a high-grade serous ovarian cancer cell model.

7. The method of claim 4, wherein, the candidate genes include ovarian cancer in vivo growth targets and ovarian cancer immune targets.

8. The method of claim 7, wherein, The ovarian cancer in vivo growth targets include at least one of HMBS, ARHGEF16, CLDN3, KMT2C, CDKN1B, CCDC6, QK, JADE1, NFE2L3 and SETD1; and the ovarian cancer immune targets include at least one of BATF, KDM2B, TARS2, PSMB10, NTRK1, ADAMTS12, HS6ST2, POLB, NID2 and FAM178B.

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