Establishment and application of m6A single-site CRISPR screening system

Abstract

The invention discloses establishment and application of an m6A single-cell CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) screening system, and provides a gRNA conformation for m6A single-cell CRISPR screening, a method for constructing a single-cell-level m6A single-site CRISPR screening system, a method for constructing a transcriptome-level m6A single-site CRISPR screening system, a method for constructing a transcriptome-level m6A single-site CRISPR screening system, a method for constructing an m6A single-site CRISPR screening The CRISPR screening system constructed by the method disclosed by the invention can be used for performing large-scale high-throughput screening on m6A loci with obviously increased methylation in colorectal cancer liver metastasis, so that the m6A loci playing a key role in colorectal cancer liver metastasis are found, and a brand new perspective is provided for tumor prevention, precise diagnosis and precise treatment.

Description

[0001] This application is application number 202311376778.8, and the invention name is a m 6 A method, device and apparatus for processing data of liver metastasis of colorectal cancer, a divisional application with a filing date of October 24, 2023. Technical Field

[0002] The present invention belongs to the field of biomedicine, and specifically relates to m 6 A. Establishment and application of single-site CRISPR screening system. Background Art

[0003] Metastasis is one of the basic biological characteristics of malignant tumors and the main cause of treatment failure and death in most cancer patients. Therefore, conducting relevant research on tumor metastasis, discovering and identifying genes that have not yet been reported to be closely related to metastasis, and clarifying their regulatory molecular networks and mechanisms of action are of great significance for discovering new tumor metastasis biomarkers and promoting molecular diagnostic research. In addition, exploring new methods for treating tumor metastasis and developing new anti-metastasis drugs have dual value for basic and clinical translational research related to tumors, and are at the forefront of biomedical research today. A large number of articles have shown that epigenetic modification elements m at the RNA level 6 A (N6-methyladenosine) has a great impact on tumor metastasis. In human cancer tissues, m 6 A regulatory protein expression is abnormal, by affecting m 6 A modification level regulates the transcriptional expression of oncogenic and tumor suppressor genes, causing tumorigenesis, proliferation, invasion, metastasis, and radiotherapy and chemotherapy resistance. However, current technical means can only 6 The influence of A methylation on tumor metastasis cannot be determined on a large scale. 6 A site methylation changes play a truly critical role in tumor metastasis.

[0004] Therefore, the development of large-scale screening m 6 A new method for single-site amplification is useful for clarifying m 6 A site and the further development of precise targeting technology for the treatment of tumor metastasis play a vital role. Summary of the invention

[0005] To make up for the shortcomings of the prior art, the present invention has developed a single-cell level m 6 A Single-site CRISPR screening system and transcriptome-level m 6 A single-site CRISPR screening system for elucidating the 6The influence of A methylation changes on the expression of downstream tumor metastasis-related target genes. Combining the above-mentioned technical means, this invention focuses on the molecular mechanism of tumor metastasis at a more microscopic level. 6 A unit point angle illustrates a single m 6 Effect of A methylation changes on tumor metastasis.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] A first aspect of the present invention provides a method for m 6 A gRNA vector for single-cell CRISPR screening, the gRNA vector comprising at least one of a CS2 sequence, a DR sequence, an SG sequence, TCGG, GGCC, an X1 sequence or an X2 sequence, wherein the CS2 sequence is a Capture Sequence 2 sequence for sequencing capture; the DR sequence is a direct repeat sequence, the X1 and X2 sequences are truncated sequences of the DR sequence, and the SG sequence is a gRNA sequence.

[0008] Furthermore, the gRNA vector also includes T*n, wherein n≥7.

[0009] Further, the gRNA vector is TCGG-CS2-TCGG-DR-SG or

[0010] The connections are made in sequence in the form of X1-GGCC-CS2-GGCC-X2-SG.

[0011] Further, the gRNA vector is sequentially connected in the manner of TCGG-CS2-TCGG-DR-SG-T*n or X1-GGCC-CS2-GGCC-X2-SG-T*n, where n≥7.

[0012] Furthermore, the gRNA vector is sequentially connected in the manner of TCGG-CS2-TCGG-DR-SG-T*7 or X1-GGCC-CS2-GGCC-X2-SG-T*7.

[0013] Furthermore, the gRNA vector is sequentially connected in the manner of X1-GGCC-CS2-GGCC-X2-SG-T*7.

[0014] Furthermore, the CS2 sequence is shown in SEQ ID NO: 1, the DR sequence is shown in SEQ ID NO: 2, the X1 sequence is shown in SEQ ID NO: 6, and the X2 sequence is shown in SEQ ID NO: 7.

[0015] The second aspect of the present invention provides a method for constructing a single cell level m 6A method for a single-site CRISPR screening system, the method comprising the following steps:

[0016] (1) Construction of stable expression m 6 A stably transfected cell line with the site-directed erasure tool;

[0017] (2) According to the target m 6 A site, construct gRNA library, package lentivirus, and infect the cell line constructed in (1);

[0018] The gRNA described in (2) is connected to the gRNA vector described in the first aspect of the present invention.

[0019] Furthermore, the MOI of lentiviral infection is less than or equal to 0.3.

[0020] Furthermore, at least 100-200 cells were infected with the same gRNA during lentiviral infection.

[0021] Furthermore, at least 100 cells were infected with the same gRNA during lentiviral infection.

[0022] Furthermore, the cell line is a tumor cell line.

[0023] Furthermore, the tumor cell lines include colorectal cancer cell lines, liver cancer cell lines, lung cancer cell lines, and gastric cancer cell lines.

[0024] Furthermore, the tumor cell line is selected from colorectal cancer cell line.

[0025] Furthermore, in step (1), m 6 A site-directed erasure tool is a fusion of the dCas protein with the catalytic domain of a demethylase.

[0026] Furthermore, the dCas protein includes dCas13 protein.

[0027] Furthermore, the dCas13 protein includes dCas13a protein, dCas13b protein, dCas13c protein, dCas13d protein, dCas13X protein, and dCas13Y protein.

[0028] Further, the dCas13 protein is selected from dCas13a protein, and the dCas13a protein is a dead Cas13a protein that has no nucleic acid cleavage activity but has the ability to bind to the target RNA under gRNA targeting.

[0029] Furthermore, the demethylase includes ALKBH5 and FTO.

[0030] Furthermore, the demethylase is selected from ALKBH5.

[0031] Furthermore, the method also includes a step of drug screening after infecting the cell line.

[0032] Furthermore, Blasticidin was used for drug screening.

[0033] Furthermore, the duration of drug screening for Blasticidin is 3-4 days.

[0034] Furthermore, the duration of drug screening for Blasticidin was 4 days.

[0035] The third aspect of the present invention provides a method for constructing a transcriptome level m 6 A method for a single-site CRISPR screening system, the method comprising:

[0036] (1) Construction of stable expression m 6 A stably transfected cell line with the site-directed erasure tool;

[0037] (2) constructing a gRNA library targeting key genes, packaging it into lentivirus, and infecting the cell line in (1);

[0038] (2) The gRNA described in step (2) is connected to the gRNA vector;

[0039] The sequences of the vector are sequentially connected in the form of DR sequence, SG sequence, and T*n, wherein n≥7.

[0040] Furthermore, the sequences of the vector are sequentially connected in the form of DR sequence, SG sequence, and T*7, wherein the DR sequence is as shown in SEQ ID NO: 2, and the SG sequence is the gRNA sequence.

[0041] Furthermore, in step (1), m 6 A site-directed erasure tool is a fusion of the dCas protein with the catalytic domain of a demethylase.

[0042] Furthermore, the dCas protein includes dCas13 protein.

[0043] Furthermore, the dCas13 protein includes dCas13a protein, dCas13b protein, dCas13c protein, dCas13d protein, dCas13X protein, and dCas13Y protein.

[0044] Further, the dCas13 protein is selected from dCas13a protein, and the dCas13a protein is a dead Cas13a protein that has no nucleic acid cleavage activity but has the ability to bind to the target RNA under gRNA targeting.

[0045] Furthermore, the demethylase includes ALKBH5 and FTO.

[0046] Furthermore, the demethylase is selected from ALKBH5.

[0047] Furthermore, in step (2), at least 100-200 cells of the infected cell line are transfected with the same gRNA.

[0048] Furthermore, the method also includes a step of drug screening after infecting the cell line.

[0049] Furthermore, Blasticidin was used for drug screening.

[0050] Furthermore, the duration of drug screening for Blasticidin is 3-4 days.

[0051] Furthermore, the duration of drug screening is 4 days.

[0052] Furthermore, the cell line is a tumor cell line.

[0053] Furthermore, the tumor cell lines include colorectal cancer cell lines, liver cancer cell lines, lung cancer cell lines, and gastric cancer cell lines.

[0054] Furthermore, the tumor cell line is selected from colorectal cancer cell line.

[0055] The fourth aspect of the present invention provides the gRNA vector described in the first aspect of the present invention in m 6 A single-cell CRISPR screening application.

[0056] The fifth aspect of the present invention provides a single cell level m constructed by the method according to the second aspect of the present invention. 6 A Single-site CRISPR screening system.

[0057] The sixth aspect of the present invention provides a transcriptome level m constructed by the method according to the third aspect of the present invention 6 A Single-site CRISPR screening system.

[0058] The seventh aspect of the present invention provides the single cell level m according to the fifth aspect of the present invention 6 A single-site CRISPR screening system or the transcriptome level m of the sixth aspect of the present invention 6 A single-site CRISPR screening system plays a key role in screening m 6 Application in site A.

[0059] The eighth aspect of the present invention provides m of SOX2 gene 6 Use of an inhibitor of A-1398 single-site methylation level in the preparation of a pharmaceutical composition for treating colorectal cancer.

[0060] Furthermore, the inhibitor includes small molecule compounds, nucleic acids, and active peptides.

[0061] Furthermore, the inhibitor is selected from nucleic acids.

[0062] Furthermore, the sequences of the nucleic acids are sequentially connected in the form of X1-GGCC-CS2-GGCC-X2-SG-T*7.

[0063] Among them, the CS2 sequence is shown as SEQ ID NO: 1, the DR sequence is shown as SEQ ID NO: 2, the X1 sequence is shown as SEQ ID NO: 6, and the X2 sequence is shown as SEQ ID NO: 7, wherein SG is a gRNA targeting the SOX2 gene, and the sequence of the gRNA is shown as SEQ ID NO: 8-12.

[0064] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0065] Advantages and beneficial effects of the present invention:

[0066] This paper proposes for the first time to identify mRNAs with significantly increased methylation in colorectal cancer liver metastasis at the transcriptome level. 6 A large-scale high-throughput screening was performed to identify mA sites that play a key role in colorectal cancer liver metastasis. 6 A site, from m 6 A single site perspective clarifies the mechanism of colorectal cancer liver metastasis and provides a new perspective for tumor prevention, diagnosis and precision treatment.

[0067] m 6 A single-cell CRISPR screening technology for m 6 A single-base editing gRNA is modified for subsequent single-cell sequencing, and then the m in each cell is sequenced using the relevant algorithm (Computer Software Copyright Registration Number 2023SR0365699). 6 A methylation change level is directly correlated with single-cell gene expression data, allowing analysis of hundreds of different m 6 A methylation editing and detection of single guide RNA (sgRNA) with direct gene expression phenotypes in hundreds to tens of thousands of cells greatly expands the operability, scalability and resolution of high-throughput functional screening at the transcriptome level. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 This is a differentially expressed gene map of colorectal cancer liver metastasis, where 1A is m 6 A is a diagram of differentially expressed genes with methylation modification, and 1B is a Venn diagram of differentially expressed genes;

[0069] Figure 2 The SOX2 gene m 6 A horizontal graph;

[0070] Figure 3 is m 6 A is a diagram of gRNA sequences and conformations in single-cell CRISPR screening, where 3A is the Capture Sequence2 sequence and location map used by gRNA, and 3B is a map of 8 gRNA vectors;

[0071] Figure 4 It is a schematic diagram of truncated X1 and X2 sequences of DR sequence;

[0072] Figure 5 is the modified gRNA conformation m 6 A horizontal graph;

[0073] Figure 6 It is the m of SOX2 gene 6 A-1398 single-site methylation inhibits tumor migration, wherein 6A is a tumor migration map, and 6B is a statistical map of tumor migration quantity. DETAILED DESCRIPTION

[0074] The definitions of some terms used in this specification are provided below. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0075] The present invention provides a method for constructing a single cell level m 6 A method for a single-site CRISPR screening system, the method comprising the following steps:

[0076] (1) Construction of stable expression m 6 A stably transfected cell line with the site-directed erasure tool;

[0077] (2) According to the target m 6 A site, construct gRNA library, package lentivirus, and infect the cell line constructed in (1);

[0078] The gRNA described in (2) is connected to the gRNA vector described in the first aspect of the present invention.

[0079] In the present invention, the method further comprises infecting the cell line constructed in (1) with the lentivirus in (2), followed by 3-4 days of blasticidin drug screening to enrich gRNA, first collecting the control group cells after the drug screening, and then leaving the cells containing gRNA, and then continuing to culture the cells for at least 10 days, collecting the cells as the experimental group, and then directly using the existing 10x Genomics to perform single-cell transcriptome sequencing, and then combining the single-cell gene expression data and the single-cell mRNA in combination with the algorithm developed by the patent co-applicant (Computer Software Work Registration No. 2023SR0365699). 6 A methylation changes can be directly correlated to the level of mRNA, allowing the analysis of hundreds of different mRNAs without knowing the cell type or markers. 6 Methylation editing of the A site.

[0080] In the present invention, CRISPR or CRISPR system refers collectively to transcripts or synthetically produced transcripts and other elements that participate in the expression of CRISPR-associated (Cas) genes or direct their activation, including sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr mate sequences (covering direct repeats and tracrRNA-processed partial direct repeats in the context of endogenous CRISPR systems), guide sequences (also called spacer sequences, gRNA in the context of endogenous CRISPR systems) or other sequences and transcripts from CRISPR loci.

[0081] In some embodiments, one or more elements of CRISPR are derived from type I, type II or type III CRISPR systems. In some embodiments, one or more elements of CRISPR are derived from specific organisms containing endogenous CRISPR, such as Streptococcus pyogenes. In general, CRISPR is characterized by promoting the formation of elements of CRISPR complexes at target sequence sites (also referred to as prototype spacer sequences in the context of endogenous CRISPR systems). In the context of forming CRISPR complexes, target sequence refers to a nucleic acid sequence to which the guide sequence is designed to have complementarity, wherein the hybridization between the target sequence and the guide sequence promotes the formation of CRISPR complexes. It is not necessary to be fully complementary, as long as there is enough complementarity to cause hybridization and promote the formation of CRISPR complexes. The target sequence can include any polynucleotide, such as DNA or RNA polynucleotides.

[0082] In some embodiments, the guide RNA (gRNA or sgRNA) can be a short, synthetic, chimeric tracrRNA / crRNA. The guide RNA can also include two short, synthetic tracrRNA / crRNAs (dual guide RNA or dgRNA).

[0083] In step (1), m 6 A site-directed erasure tool is a fusion of the dCas protein with the catalytic domain of a demethylase.

[0084] The dCas protein includes dCas13 protein.

[0085] The dCas13 protein includes dCas13a protein, dCas13b protein, dCas13c protein, dCas13d protein, dCas13X protein (also called Cas13e), and dCas13Y protein (also called Cas13f).

[0086] Further, the dCas13 protein is selected from dCas13a protein, and the dCas13a protein is a dead Cas13a protein that has no nucleic acid cleavage activity but has the ability to bind to the target RNA under gRNA targeting.

[0087] In some embodiments, the Cas 13a protein is or is derived from the following species: Bacteroides, Blautia, Butyrivibrio, Carnobacterium, Chloroflexus, Clostridium, Demequina, Eubacterium, Herbinix, Insolitispirillum, Lachnospiraceae, Leptotrichia, Listeria, Paludibacter, Porphyromonadaceae, Pseudobutyrivibrio, Rhodobacter or Thalassospira; preferably, Leptotrichia shahii, Listeria seeligeri), Lachnospiraceae bacterium (such as Lb MA2020, LbNK4A179, Lb NK4A144), Clostridium aminophilum (such as Ca DSM 10710), Carnobacterium gallinarum (such as Cg DSM 4847), Paludibacterpropionicigenes (such as Pp WB4), Listeriaweihenstephanensis (such as Lw FSL R9-0317), Listeriaceaebacterium (such as Lb FSL M6-0635), Leptotrichia wadei (such as Lw F0279), Rhodobacter capsulatus (such as Rc SB 1003, Rc R121, Rc DE442), Leptotrichia buccalis (such as Lb C-1013-b), Herbinix hemicellulosilytica, Eubacteriaceae bacterium (such as Eb CHKCI004), Blautia sp.) Marseille-P2398, Leptotrichia sp. oral taxon 879str.F0557, Chloroflexus aggregans, Demequina aurantiaca, Thalassospira sp. TSL5-1, Pseudobutyrivibrio s p. OR37, Butyrivibrio sp. YAB3001, Leptotrichia sp. Marseille-P3007, Bacteroid es ihua e, Porphyromonadaceae bacterium (such as PbKH3CP3RA), Listeria riparia or Insolitispirillum peregrinum. .

[0088] In the present invention, m 6 A or m6A can also be called N 6 -Methyladenine or N6-methyladenosine.

[0089] In the present invention, demethylase refers to m 6 A demethylases, including but not limited to FTO and ALKBH5.

[0090] In a specific embodiment of the present invention, the demethylase is selected from ALKBH5.

[0091] The cell line is a tumor cell line.

[0092] In the present invention, a tumor is a new organism formed by the proliferation and abnormal differentiation of body cells under the action of various initiating and promoting factors. The growth of a tumor is not regulated by normal body physiology, but destroys normal tissues and organs.

[0093] In the present invention, tumors include but are not limited to adrenocortical carcinoma, adult cancer of unknown primary site, adult malignant mesothelioma, AIDS-related cancer, anal cancer, appendix cancer, astrocytoma (cerebellar astrocytoma, cerebral astrocytoma, childhood cerebral astrocytoma, pineal astrocytoma), basal cell carcinoma, bile duct cancer, bladder cancer, bone tumor, brain cancer, breast cancer, bronchial adenoma / carcinoid, carcinoid tumor, cancer of unknown primary site, cervical cancer, chronic myeloproliferative disorder, colon cancer, desmoplastic small round cell tumor, endometrial cancer, ependymoma, epithelioid hemangioendothelioma (EHE), food Tumors, Ewing's sarcoma family of tumors, extracranial germ cell tumors, extragonadal germ cell tumors, eye cancer, gallbladder cancer, gastrointestinal stromal tumors (GIST), gestational trophoblastic tumors, gliomas, colorectal cancer, head and neck cancer, heart cancer, liver cancer, islet cell carcinoma (endocrine pancreas), Kaposi's sarcoma, kidney cancer (renal cell carcinoma), laryngeal cancer, leukemias (acute lymphoblastic leukemia, chronic lymphocytic leukemia (also known as chronic lymphocytic leukemia), chronic myeloid leukemia (also known as chronic myeloid leukemia), hairy cell leukemia), lip cancer, liposarcoma, lung cancer (non-small cell lung cancer, small cell lung cancer) Lung cancer), lymphoma (non-Hodgkin lymphoma, Hodgkin lymphoma, AIDS-related lymphoma, Burkitt lymphoma, central nervous system lymphoma, cutaneous T-cell lymphoma), macroglobulinemia, malignant fibrous histiocytoma of bone / osteosarcoma, medulloblastoma, melanoma, Merkel cell carcinoma, squamous cell carcinoma of the neck with occult metastatic primary, multiple endocrine neoplasms syndrome, bone marrow cancer, multiple myeloma / plasma cell neoplasms, mycosis fungoides, myelodysplastic syndrome, myxoma, nasal and paranasal sinus cancer, neuroblastoma, ovarian cancer, ovarian epithelial carcinoma (surface epithelial stromal tumor), ovarian fibrosis, ... Germinoma, ovarian low malignant potential tumor, pancreatic cancer, parathyroid cancer, penile cancer, pharyngeal cancer (hypopharynx cancer, nasopharyngeal cancer, oropharyngeal cancer), pheochromocytoma, pineal germ cell tumor, pineoblastoma, supratentorial primitive neuroectodermal tumor, pituitary adenoma, pleuropulmonary blastoma, prostate cancer, colorectal cancer, transitional cell carcinoma of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Sézary syndrome, Merkel cell skin cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, testicular cancer, thymoma, thyroid cancer, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer.

[0094] In a preferred embodiment of the present invention, the tumor includes colorectal cancer, liver cancer, lung cancer, and gastric cancer.

[0095] In a specific embodiment of the invention, the tumor is selected from colorectal cancer.

[0096] The present invention provides a method for constructing a transcriptome-level m 6A method for a single-site CRISPR screening system, characterized in that the method comprises:

[0097] (1) Construction of stable expression m 6 A stably transfected cell line with the site-directed erasure tool;

[0098] (2) constructing a gRNA library targeting key genes, packaging it into lentivirus, and infecting the cell line in (1);

[0099] The sequences of the vector are sequentially connected in the form of DR sequence, SG sequence, and T*n, wherein n≥7.

[0100] Furthermore, the sequences of the vector are sequentially connected in the form of DR sequence, SG sequence, and T*n, wherein n is 7.

[0101] In the present invention, the method also includes enriching gRNA through 3-4 days of blasticidin drug screening after infecting the cell line in (1). The cells left after the drug screening are the cells containing gRNA. First, the control group cells are collected, and then they are treated with oxaliplatin for the treatment of colorectal cancer for screening. The surviving cells are compared and high-throughput sequencing (HighThroughputSequencing) is also called next generation sequencing (Next Generation Sequencing, NGS). If the cell survival is more than the control group, see which genes are enriched after methylation is removed to resist oxaliplatin; if the cell survival is less than the control, then it is the target after gene demethylation that helps oxaliplatin to exert its efficacy. Through the colorectal cancer liver metastasis model, the transcriptome level m 6 A methylation high-throughput screening system.

[0102] The present invention provides m of SOX2 gene 6 Use of an inhibitor of A-1398 single-site methylation level in the preparation of a product for treating colorectal cancer.

[0103] In the present invention, m of SOX2 gene 6 A-1398 single-site methylation inhibitors include m-SOX2 6 A-1398 single-site methylation levels of small molecule compounds, nucleic acids, and active peptides.

[0104] In the present invention, the pharmaceutical composition can be a dosage form that is convenient for administration prepared by conventional methods, including but not limited to aqueous solution injection, powder injection, pills, powders, tablets, patches, suppositories, emulsions, creams, gels, granules, capsules, aerosols, sprays, powder sprays, sustained-release agents and controlled-release agents.

[0105] The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0106] In the present invention, pharmaceutically acceptable carriers may include inert ingredients that do not unduly inhibit the biological activity of the compound. Pharmaceutically acceptable carriers should be biocompatible, for example, non-toxic, non-inflammatory, non-immunogenic or non-other undesirable reactions or side effects when administered to a subject. Standard pharmaceutical preparation techniques can be used.

[0107] Some examples of substances that can serve as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffer substances (such as tween 80, phosphates, glycine, sorbic acid or potassium sorbate), partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride or zinc salts), colloidal silicon dioxide, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, methylcellulose, hydroxypropyl methylcellulose, lanolin; sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as Sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol or polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol, and phosphate buffered solutions, and other nontoxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, and colorants, release agents, coating agents, sweeteners, flavoring agents and perfuming agents, preservatives and antioxidants may also be present in the composition, according to the judgment of the formulator.

[0108] Any oral acceptable dosage form (including but not limited to capsules, tablets, aqueous suspensions or solutions) can be used for oral administration. For tablets for oral administration, commonly used carriers include lactose and corn starch. Lubricants such as magnesium stearate are typically also added. For oral administration in capsule form, available diluents include lactose and dry corn starch. When an aqueous suspension is required for oral administration, the active ingredient is combined with an emulsifier and a suspending agent. If necessary, certain sweeteners, flavorings or coloring agents may also be added.

[0109] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compound, the liquid dosage form may also contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, oral compositions may also include adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and aromatics.

[0110] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound is mixed with at least one inert pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and / or a) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol and silicic acid, b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and gum arabic, c) humectants such as glycerol, d) disintegrants such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate, e) solution retardants such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as cetyl alcohol and glyceryl monostearate, h) absorbents such as kaolin and bentonite, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

[0111] Solid compositions of similar type may also be used as fillers in soft and hard filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols, etc. Solid dosage forms of tablets, lozenges, capsules, pills and granules may be prepared with coatings and shells, such as enteric coatings and other coatings well known in the field of pharmaceutical formulations. They may optionally contain opacifiers and may also have a composition that allows them to release the active ingredient only or preferentially in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymers and waxes. Solid compositions of similar type may also be used as fillers in soft and hard filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols, etc.

[0112] Microencapsulated forms with one or more of the above excipients can also be used in the present invention. Solid dosage forms of tablets, lozenges, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings, controlled release coatings and other coatings well known in the field of pharmaceutical formulation. In such solid dosage forms, the active compound can be mixed with at least one inert diluent such as sucrose, lactose or starch. As is common practice, such dosage forms can also contain additional substances that are not inert diluents, such as tableting lubricants and other tableting aids such as magnesium stearate and microcrystalline cellulose. For capsules, tablets and pills, the dosage form can also include a buffer. They can optionally contain an opacifier and can also have a composition that allows them to release the active ingredient only or preferentially in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymers and waxes.

[0113] The invention will be further described below in conjunction with specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the invention. The main features of the invention may be used in a variety of embodiments without departing from the scope of the invention.

[0114] Example 1 m at the transcriptome level 6 A Single-site CRISPR Screening System

[0115] 1 Experimental Materials

[0116] 1.1 Cell culture

[0117] (1) Colorectal cancer cell line HCT116 (Wuhan Prosac Life Science Co., Ltd., CL-0096)

[0118] (2) McCoy 5A medium (SH30200.01, HyClone)

[0119] (3)0.25% Trypsin-EDTA (Thermofisher, 25200072)

[0120] (4) Reduced serum medium Opti-MEM TM (Thermofisher, 31985070)

[0121] (5)Doxycycline hyclate(sigma,D9891)

[0122] (6) Puromysin (Selleck, S7417)

[0123] 1.2 Immunofluorescence

[0124] (7) 4% paraformaldehyde (Meilunbio, MA0192)

[0125] (8) PBS (Hyclone, SH30256.01)

[0126] (9)Triton X-100 (Sigma, T8787)

[0127] (10)Bovine Serum Albumin (BSA) (A1933, sigma)

[0128] (11) DAPI (sigma, D9542)

[0129] (12) Immunofluorescence primary antibody: HA-Tag (Proteintech, 51064-2-AP)

[0130] (13) Immunofluorescence secondary antibody: Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 (A-31572, Thermofisher)

[0131] 1.3 Western blot

[0132] (14) Protein lysis buffer: RIPA Buffer (10×) (9806, CST), Protease Inhibitor Cocktail (MCE, HY-K0010)

[0133] (15) Protein Marker (Thermofisher, 26619)

[0134] (16) BCA protein concentration assay kit (enhanced) (Beyotime, P0010)

[0135] (17) SDS-PAGE gel preparation kit (Beyotime, P0012A)

[0136] (18) Western blot primary antibody: HA-Tag (66006-1-Ig, Proteintech)

[0137] (19) Western blot secondary antibody: horseradish peroxidase-labeled goat anti-mouse IgG (H+L) (A0216, Beyotime)

[0138] 1.4SELECT detection reagent

[0139] (20)CutSmart(10×)(B7204S, NEB)

[0140] (21)Bst 2.0DNA Polymerase (M0537S, NEB)

[0141] (twenty two) Ligase (M0375S, NEB)

[0142] (23) 5'-Adenosine triphosphate (ATP) (P0756S, NEB)

[0143] 2 Experimental methods and results

[0144] 2.1 In the colorectal cancer HCT116 cell line, stably expressing m 6 A Stable cell line for site-directed erasure tools

[0145] 1) The well-growing colorectal cancer cell line HCT116 (Wuhan Pronocell Biotechnology Co., Ltd., CL-0096) was digested into single cells with 0.25% Trypsin-EDTA (thermofisher, 25200072) and cultured in reduced serum medium Opti-MEM TM (thermofisher, 31985070) resuspend and count;

[0146] 2) Use the CUY21 EDIT II electroporator from BEX. Add 1 μg m 6 A site-directed erasure tool plasmid (or negative control plasmid) and 100 ng of transposase plasmid were electroporated;

[0147] 3) The electroporated HCT116 cells were seeded in a six-well plate and cultured in McCoy 5A medium for 2 days. Subsequently, the medium was replaced with McCoy 5A medium containing Doxycycline hyclate (Dox) and puromysin for drug screening for 7 to 14 days until positive cell clones appeared.

[0148] 2.2 Validation of mRNA expression in colorectal cancer cell lines using immunofluorescence and Western blot 6 A Fixed-point erase tool expression

[0149] 1) Immunofluorescence: Fixed cell samples: The positive cells after drug screening were passaged at a ratio of 1:2. After 24 hours of attachment, one well was not induced with Dox, and the other well was induced with Dox. After 48 hours, the cells were fixed and fixed with 4% paraformaldehyde at room temperature for 15 minutes, and washed three times with PBS; Permeabilization: Permeabilization with PBS diluent containing 0.3% Triton-X 100 at room temperature for 30 minutes, and washed three times with PBS solution; Blocking: After blocking with 3% BSA blocking solution for 1 hour, add primary antibody and incubate overnight at 4°C or at room temperature for 2 hours; Apply secondary antibody: Wash with PBS solution three times, add fluorescent secondary antibody, and incubate at room temperature in the dark for 1 hour. Statistics of experimental results: Wash with PBS solution three times, incubate with DAPI at room temperature in the dark for 5 minutes, and observe and take pictures with laser confocal microscope or fluorescence microscope after washing with PBS solution.

[0150] 2) Western blot: Protein sample preparation: The positive cells after drug screening were subcultured at a ratio of 1:2. After 24 hours of attachment, one well was not induced with Dox, while the other well was induced with Dox. After another 48 hours, the cells were collected and an appropriate amount of protein lysis buffer was added. The cells were placed on ice for 30 minutes for full lysis. The cells were centrifuged at 12,000 rpm / min and 4°C for 2 minutes. The supernatant was transferred to a new EP tube and placed on ice. Protein quantification: The extracted protein samples were quantified using a BCA kit. The volume required for 30 μg of total protein in the sample was calculated. The final volume was 30 μL. 5 μL of 6× loading Buffer, calculate the volume of ddH2O to be added, add water and loading Buffer first, then add protein, mix well by pipetting, boil in a 95℃ metal bath for 10 min, and place on ice for later use; prepare SDS-PAGE gel: prepare SDS-PAGE gel with a separation gel concentration of 8% and a concentration gel with a concentration of 5%, insert the sample well comb, and wait for solidification; load the sample: take out the prepared SDS-PAGE gel, assemble the electrophoresis tank, remove the sample well comb, add 1×SDS-PAGE electrophoresis buffer, load 30μg protein per well, and add 8μL marker to one well at each end. To avoid edge effects, it is best to use the middle hole for sample injection; electrophoresis is performed at a constant voltage of 80V until the sample reaches the bottom of the concentrated gel to form a straight line, and the voltage is adjusted to 120V for separation; 30 minutes before the end of electrophoresis, the PVDF membrane is immersed in methanol for 15 seconds for activation, and the membrane changes from opaque to translucent without white spots, and then the membrane is completely immersed in the transfer buffer for equilibrium for more than 5 minutes, and the sponge and filter paper are immersed in the transfer buffer at the same time; transfer: after the electrophoresis is completed, the gel, sponge, filter paper and PVDF membrane are immersed in the buffer to prepare a "sponge-filter paper-gel-membrane-filter paper-sponge" sandwich and try to remove bubbles, put the sandwich into the transfer device, bury the device in ice, and transfer the membrane at a constant current of 200-250mA for 60- 90min; Blocking: After the transfer, the PVDF membrane was taken out, rinsed slightly in 1×TBST, immersed in blocking solution, and blocked slowly at room temperature for 1h; Incubation with primary antibody: Cut the membrane into appropriate size according to the molecular weight of the target protein and the internal reference protein, immerse it in the primary antibody incubation solution (diluted with blocking solution), and incubate it in a 4°C shaker with slow shaking overnight; Post-primary antibody membrane washing: After overnight incubation with primary antibody, wash it three times with 1×TBST with shaking, each time for 5min; Incubation with secondary antibody: Immerse the membrane in the secondary antibody incubation diluted with blocking solution, and incubate it slowly with shaking at room temperature for 1h; Post-secondary antibody membrane washing: After incubation for 1h, wash it three times with 1×TBST with shaking, each time for 5min; Add chemiluminescent liquid to the membrane, and expose the protein bands in the chemiluminescence instrument for analysis.

[0151] 2.3 Identify potential key genes affecting colorectal cancer liver metastasis

[0152] (1) Two groups of paired clinical sample data of colorectal cancer in situ lesions and liver metastases were collected from the Department of Clinical Pathology for MeRIP-seq and RNA-seq sequencing. After joint analysis, 395 liver metastases were found to have m 6 A Genes with significant differences in methylation modification and expression levels ( Figure 1 A).

[0153] (2) Bioinformatics analysis was performed using the colorectal cancer liver metastasis public database GSE50760, and 3162 differentially expressed genes were found (P < 0.05, |log2FC| > 1). A Wayne analysis was performed with the self-test data, and a total of 84 common genes were found ( Figure 1 B) These 84 genes were defined as potential key genes affecting colorectal cancer liver metastasis.

[0154] 2.4 Validation of m by SELECT in the colorectal cancer system 6 A site-directed eraser plays a role in single-site demethylation

[0155] (1) Based on previous work, relevant literature showed that SOX2 is a stemness gene in colorectal cancer cell lines and is expressed in the liver after liver metastasis. 6 m at A-1398 6 A methylation level was significantly increased, so the SOX2 gene was selected as the test gene.

[0156] (2) Five sgRNAs were designed for this site, S1 was located upstream of signal A1398, 33 nt away; S2 and S3 covered signal A1398; S4 and S5 were located downstream of signal A1398, 3 nt and 203 nt away, respectively. In order to evaluate the possibility of crRNA off-target, non-targeting crRNA (NT) was used as a negative control. Stable cell lines containing different crRNAs were treated with Dox induction, and the m 6 The results showed that only under the targeting of S1 and S3, the amount of SELECT products increased significantly compared with the control group, with statistical differences, indicating that after Dox induction, the m 6 The A level decreased significantly, and the experimental results showed that TRME editor can not only cause specific m 6 The methylation level of site A was reduced and there was a very precise editing window, indicating that a single-site demethylation system was successfully constructed in the colorectal cancer HCT116 cell line, which can be used for subsequent screening work (S1-5 represents sgRNA1-5, Figure 2 , Table 1).

[0157] Table 1 sgRNA (gRNA) sequences

[0158]

[0159]

[0160] 2.5 Construction of gRNA library and packaging of lentivirus

[0161] Based on the 84 potential key genes that affect colorectal cancer liver metastasis screened above, the possible m 6 Site A and the gRNA sequence targeting the above site. The patent applicant handed over the gRNA sequence to Suzhou Jinweizhi Biotechnology Co., Ltd. for chip synthesis, and further constructed it on the gRNA vector used in the early stage. In order to ensure that most cells are transferred with only one gRNA, rather than multiple gRNAs at the same time, it is necessary to construct a lentivirus with a higher transfection efficiency and an MOI of less than or equal to 0.3. This part of the lentivirus packaging was completed by Suzhou Anshengda Company.

[0162] 2.6gRNA library infection and stable expression 6 A Stable cell line for site-directed erasure tools

[0163] (1) Using synthetic lentivirus to stably express m 6 A: For the stable transfection cell line of the site-directed erasure tool, to ensure the transfection efficiency, at least 100 to 200 cells should be transfected with the same gRNA.

[0164] (2) Since the gRNA vector carries the blasticidin resistance gene, after the gRNA is transfected into the cells, the cells are screened with blasticidin for 4 days, and the surviving cells are transfected with the gRNA library.

[0165] (3) The cells before drug screening were collected as the control group, and then treated with oxaliplatin (20uM) for the treatment of colorectal cancer for screening. The surviving cells were collected as the experimental group. The genomes of the control group and experimental group cells were extracted and sent to Suzhou Genewise Biotechnology Co., Ltd. for NSG sequencing and data analysis.

[0166] Example 2 Single cell level 6 A Single-site CRISPR Screening System

[0167] 1 Experimental Materials

[0168] 1.1 Cell culture

[0169] (1) Colorectal cancer cell line HCT116 (Wuhan Prosac Life Science Co., Ltd., CL-0096)

[0170] (2) McCoy 5A medium (SH30200.01, HyClone)

[0171] (3)0.25% Trypsin-EDTA (Thermofisher, 25200072)

[0172] (4) Reduced serum medium Opti-MEM TM (Thermofisher, 31985070)

[0173] (5)Doxycycline hyclate(sigma,D9891)

[0174] (6) Puromysin (Selleck, S7417)

[0175] (7) Blasticidin S HCl (Selleck, S7419)

[0176] 1.2 Homologous recombination

[0177] (8) MultiS One Step Cloning Kit (C113-02, Vazyme)

[0178] 1.3SELECT detection reagent

[0179] (9)CutSmart(10×)(B7204S, NEB)

[0180] (10)Bst 2.0DNA Polymerase (M0537S, NEB)

[0181] (11) Ligase (M0375S, NEB)

[0182] (12) Adenosine 5'-triphosphate (ATP) (P0756S, NEB)

[0183] 1.4 Transwell experiment

[0184] (13) Polycarbonate membrane cell culture inserts (chambers) (CLS3422, Sigma)

[0185] 2 Experimental methods and results

[0186] 2.1 In the colorectal cancer HCT116 cell line, stably expressing m 6 A Stable cell line of the targeted erasure tool (same method as in Example 1)

[0187] 2.2 Validation of mRNA expression in colorectal cancer cell lines using immunofluorescence and Western blot 6 A fixed-point erasing tool expression (method is the same as in Example 1)

[0188] 2.3 Modification of gRNA vectors for single-cell sequencing

[0189] (1) The patent applicant has developed a novel CaptureSequence2 sequence and location based on the gRNA currently used in single-cell CRISPR screening ( Figure 3 A), 8 conformations were designed (gRNA vector, Figure 3 B, Table 2), and the single-site demethylated gRNA vector was transformed by homologous recombination. According to the sequencing results, the transformation was successful, wherein the X1 sequence or the X2 sequence is a truncated sequence of the DR sequence, and its truncated form is as follows Figure 4 shown.

[0190] Table 2 8 gRNA vector sequences

[0191]

[0192]

[0193] 2.4 In the colorectal cancer system, the ability of modified gRNA to demethylate at a single site was verified by the SELECT method

[0194] (1) In this part of the verification work, the SOX2 gene m 6 The A-1398 site was selected as the methylation target site, and S1 with the strongest single-site demethylation was selected as the gRNA sequence. Eight gRNA vectors (numbered 1 to 8) containing the same gRNA sequence but with different conformations were constructed, and then packaged into viruses to stably express m 6 A stable cell line of the site-directed erasure tool. Among them, the gRNA sequence before transformation was selected as the positive control and marked as S1, and NT was selected as the negative control.

[0195] (2) Use the SELECT method to detect, such as Figure 5 As shown in the figure, it was found that only under the targeting of gRNA conformations numbered 4 and 6, the amount of SELECT products increased significantly compared with the control group. 6 The A level decreased significantly, with statistical significance, indicating that the two modified gRNA conformations still retained the ability of single-site demethylation.

[0196] (3) The two stably transfected cell lines were subjected to transwell experiments, such as Figure 6, and found that only the conformation numbered 6 inhibited tumor migration. In addition, the patent applicant confirmed for the first time that the m of the SOX2 gene 6 A-1398 single-site methylation reduction can inhibit tumor migration, so this modified gRNA was selected for subsequent single-cell m 6 A. Unit site screening.

[0197] 2.4 Identify potential genes and their corresponding m that affect the occurrence and development of colorectal cancer 6 A site

[0198] Based on the third-generation sequencing data related to the occurrence and development of colorectal cancer, the m 6 A site.

[0199] 2.5 Construction of gRNA library and packaging of lentivirus

[0200] (1) Design gRNA according to cas13design: A flexible tool to design Cas13d guide RNAs website (https: / / cas13design.nygenome.org). 6 Three gRNAs are designed at site A, and then the gRNA sequences are sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for synthesis chip construction, which is then built into a gRNA vector numbered 6 to form a gRNA library, which is packaged into a lentivirus. Similarly, to ensure that most cells are only transferred with one gRNA, rather than multiple gRNAs at the same time, it is necessary to construct a lentivirus with a higher transfection efficiency and an MOI of less than or equal to 0.3. The patent applicant entrusted the lentivirus packaging to Suzhou Anshengda Company.

[0201] 2.6gRNA library infection and stable expression 6 A Stable cell line for site-directed erasure tools

[0202] (1) After synthesizing lentivirus, infect the stable expression m 6 A: For the stable transfection cell line of the site-directed erasure tool, to ensure the transfection efficiency, at least 100 to 200 cells should be transfected with the same gRNA.

[0203] Table 3 Infection of stable cell lines

[0204]

[0205]

[0206] (2) Since the gRNA vector carries the blasticidin resistance gene, after the gRNA is transfected into the cells, the cells are screened with blasticidin for 4 days, and the surviving cells are transfected with the gRNA library.

[0207] (3) After drug screening, the control group cells are first collected, and then the cells containing gRNA are left. The cells are then cultured for at least 10 days, and the cells are collected as the experimental group. They are sent to the company to directly use the existing 10x Genomics for single-cell transcriptome sequencing, and then combined with the algorithm developed by the patent co-applicant (Computer Software Copyright Registration No. 2023SR0365699) to combine the single-cell gene expression data and the mRNA in the single cell. 6 A methylation change level is directly correlated.

[0208] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A method for m 6 A gRNA vector for single-cell CRISPR screening, characterized in that The gRNA vector comprises at least one of a CS2 sequence, a DR sequence, an SG sequence, TCGG, GGCC, an X1 sequence or an X2 sequence, wherein the X1 sequence or the X2 sequence is a truncated sequence of the DR sequence, and the SG sequence is a gRNA sequence; Preferably, the gRNA vector further comprises T*n, wherein n≥7; Preferably, the gRNA vector is sequentially connected in the form of TCGG-CS2-TCGG-DR-SG or X1-GGCC-CS2-GGCC-X2-SG; Preferably, the gRNA vector is sequentially connected in the manner of TCGG-CS2-TCGG-DR-SG-T*n or X1-GGCC-CS2-GGCC-X2-SG-T*n, n≥7; Preferably, the gRNA vector is sequentially connected in the manner of TCGG-CS2-TCGG-DR-SG-T*7 or X1-GGCC-CS2-GGCC-X2-SG-T*7; Preferably, the gRNA vector is sequentially connected in the form of X1-GGCC-CS2-GGCC-X2-SG-T*7; Preferably, the CS2 sequence is shown as SEQ ID NO: 1, the DR sequence is shown as SEQ ID NO: 2, the X1 sequence is shown as SEQ ID NO: 6, and the X2 sequence is shown as SEQ ID NO:

7.

2. A method to construct single-cell level m 6 A method of a single-site CRISPR screening system, characterized in that The method comprises the following steps: (1) Construction of stable expression m 6 A stably transfected cell line with the site-directed erasure tool; (2) According to the target m 6 A site, construct gRNA library, package lentivirus, and infect the cell line constructed in (1); The gRNA described in (2) is connected to the gRNA vector described in claim 1; Preferably, the MOI of lentiviral infection is less than or equal to 0.3; Preferably, at least 100-200 cells are infected with the same gRNA during lentiviral infection; Preferably, at least 100 cells are infected with the same gRNA during lentiviral infection; Preferably, the cell line is a tumor cell line; Preferably, the tumor cell line includes a colorectal cancer cell line, a liver cancer cell line, a lung cancer cell line, and a gastric cancer cell line; Preferably, the tumor cell line is selected from colorectal cancer cell line.

3. The method according to claim 2, characterized in that In step (1), m 6 A site-directed erasure tool refers to the fusion of the dCas protein with the catalytic domain of the demethylase; Preferably, the dCas protein includes dCas13 protein; Preferably, the dCas13 protein includes dCas13a protein, dCas13b protein, dCas13c protein, dCas13d protein, dCas13X protein, and dCas13Y protein; Preferably, the dCas13 protein is selected from dCas13a protein; Preferably, the demethylase includes ALKBH5 and FTO; Preferably, the demethylase is selected from ALKBH5.

4. The method according to claim 2, characterized in that: The method further comprises the step of drug screening after infection of the cell line; Preferably, Blasticidin is used for drug screening; Preferably, the duration of the Blasticidin drug screening is 3-4 days; Preferably, the duration of the Blasticidin drug screening is 4 days.

5. A method for constructing transcriptome-level m 6 A method of a single-site CRISPR screening system, characterized in that The method comprises: (1) Construction of stable expression m 6 A stably transfected cell line with the site-directed erasure tool; (2) constructing a gRNA library targeting key genes, packaging it into lentivirus, and infecting the cell line in (1); (2) The gRNA described in is connected to the gRNA vector; The sequences of the vector are sequentially connected in the form of DR sequence, SG sequence, T*n, wherein n≥7; Preferably, the sequences of the vector are sequentially connected in the form of DR sequence, SG sequence, and T*7, wherein the DR sequence is as shown in SEQ ID NO: 2, and the SG sequence is the gRNA sequence; Preferably, in step (1), m 6 A site-directed erasure tool refers to the fusion of the dCas protein with the catalytic domain of the demethylase; Preferably, the dCas protein includes dCas13 protein; Preferably, the dCas13 protein includes dCas13a protein, dCas13b protein, dCas13c protein, dCas13d protein, dCas13X protein, and dCas13Y protein; Preferably, the dCas13 protein is selected from dCas13a protein; Preferably, the demethylase includes ALKBH5 and FTO; Preferably, the demethylase is selected from ALKBH5; Preferably, in step (2), at least 100-200 cells of the infected cell line are transfected with the same gRNA; Preferably, the method further comprises the step of drug screening after infecting the cell line; Preferably, Blasticidin is used for drug screening; Preferably, the duration of the Blasticidin drug screening is 3-4 days; Preferably, the drug screening time is 4 days; Preferably, the cell line is a tumor cell line; Preferably, the tumor cell line includes a colorectal cancer cell line, a liver cancer cell line, a lung cancer cell line, and a gastric cancer cell line; Preferably, the tumor cell line is selected from colorectal cancer cell line.

6. The gRNA vector according to claim 1 is in m 6 A. Application of single-cell CRISPR screening.

7. The single cell level m constructed by the method according to any one of claims 2 to 4 6 A Single-site CRISPR screening system.

8. The transcriptome level constructed by the method of claim 5 6 A Single-site CRISPR screening system.

9. The single cell level m according to claim 7 6 A single-site CRISPR screening system or the transcriptome-wide screening system of claim 8 6 A single-site CRISPR screening system plays a key role in screening m 6 Application in site A.

10. SOX2 gene m 6 Use of an inhibitor of A-1398 single-site methylation level in the preparation of a pharmaceutical composition for treating colorectal cancer; Preferably, the inhibitor includes small molecule compounds, nucleic acids, and active peptides; Preferably, the inhibitor is selected from nucleic acids; Preferably, the sequences of the nucleic acids are sequentially connected in the form of X1-GGCC-CS2-GGCC-X2-SG-T*7; in, The CS2 sequence is shown in SEQ ID NO: 1, the DR sequence is shown in SEQ ID NO: 2, the X1 sequence is shown in SEQ ID NO: 6, and the X2 sequence is shown in SEQ ID NO: 7, wherein SG is a gRNA targeting the SOX2 gene, and the sequence of the gRNA is shown in SEQ ID NOs: 8-12; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

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