m 6 Establishment and Application of a Single-Locus CRISPR Screening System

Abstract

The present invention discloses the establishment and application of an m6A single-site CRISPR screening system, provides a gRNA conformation for m6A single-cell CRISPR screening, a method for constructing a single-cell level m6A single-site CRISPR screening system, and a method for constructing a transcriptome level m6A single-site CRISPR screening system. The CRISPR screening system constructed by the method of the present invention can perform large-scale high-throughput screening on m6A sites with significantly increased methylation in colorectal cancer liver metastasis, and then search for m6A sites that play a key role in colorectal cancer liver metastasis, providing a new perspective for tumor prevention, precise diagnosis and precise treatment.

Description

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

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

[0003] Metastasis is one of the basic biological characteristics of malignant tumors and is the main cause of treatment failure and death in most cancer patients. Therefore, conducting research on tumor metastasis, discovering and identifying genes that have not been reported to be closely related to metastasis and clarifying the molecular networks and mechanisms of their regulation are of great significance for discovering new tumor metastasis biomarkers and promoting molecular diagnosis research. Moreover, exploring new methods for treating tumor metastasis and developing new anti-metastatic drugs have dual value for basic and clinical translational research related to tumors and are at the forefront of current biomedical research. A large number of articles have shown that the epigenetic modification element m 6 A (N6-methyladenosine) has a great impact on tumor metastasis. In human cancer tissues, the expression of m 6 A regulatory proteins is abnormal, and by affecting the m 6 A modification level, it regulates the transcriptional expression of oncogenes and tumor suppressor genes, resulting in tumorigenesis, proliferation, invasion, metastasis, and chemoradiotherapy resistance. However, current technical means can only study the impact on tumor metastasis from the overall level of m 6 A methylation, and it is impossible to clearly identify on a large scale which specific m 6 A site methylation changes play a truly key role in tumor metastasis.

[0004] Therefore, developing a new method for large-scale screening of m 6 A single sites is crucial for identifying the m 6 A sites that actually play a key role in tumor metastasis and further developing precision targeting technologies for treating tumor metastasis. Summary of the Invention

[0005] To make up for the deficiencies of the prior art, the present invention has developed a single-cell level m 6 A single-site CRISPR screening system and a transcriptome level m 6 A single-site CRISPR screening system for clarifying individual m 6The effect of methylation changes on the expression levels of downstream target genes related to tumor metastasis. Combining the above-developed technical means, the present invention focuses the molecular mechanism of tumor metastasis on a more microscopic level, starting from the 6 single-site angle of m 6 A to clarify the effect of single-site m

[0006] A methylation changes on tumor metastasis.

[0007] To achieve the above object, the present invention adopts the following technical solutions: 6 A gRNA vector for single-cell CRISPR screening of m 1 A, the gRNA vector includes at least one of the CS2 sequence, DR sequence, SG sequence, TCGG, GGCC, X 2 sequence or X 1 sequence, wherein the CS2 sequence is the Capture Sequence 2 sequence for sequencing capture; the DR sequence is the direct repeat sequence, and X 2 sequence, X

[0008] sequence are truncated sequences of the DR sequence, and the SG sequence is the gRNA sequence.

[0009] Further, the gRNA vector further includes T*n, wherein n≥7.

[0010] Further, the gRNA vector is sequentially connected in the manner of TCGG-CS2-TCGG-DR-SG or 1 X 2 -GGCC-CS2-GGCC-X

[0011] -SG. 1 Further, the gRNA vector is sequentially connected in the manner of TCGG-CS2-TCGG-DR-SG-T*n or X 2 -GGCC-CS2-GGCC-X

[0012] -SG-T*n, n≥7. 1 Further, the gRNA vector is sequentially connected in the manner of TCGG-CS2-TCGG-DR-SG-T*7 or X 2 -GGCC-CS2-GGCC-X

[0013] -SG-T*7. 1 Further, the gRNA vector is sequentially connected in the manner of X 2 -GGCC-CS2-GGCC-X

[0014] Further, the CS2 sequence is as shown in SEQ ID NO: 1, the DR sequence is as shown in SEQ ID NO: 2, and the X 1 sequence is as shown in SEQID NO: 6, and the X 2 sequence is as shown in SEQ ID NO: 7.

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

[0016] (1) Construct a stable cell line stably expressing the m 6 A site-directed erasure tool;

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

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

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

[0020] Further, when infecting with lentivirus, at least 100-200 cells are infected with the same gRNA.

[0021] Further, when infecting with lentivirus, at least 100 cells are infected with the same gRNA.

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

[0023] Further, 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.

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

[0025] Further, in step (1), the m 6 A site-directed erasure tool refers to the fusion of the dCas protein and the catalytic domain of the demethylase.

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

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

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

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

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

[0031] Further, the method further includes the step of drug screening after infecting the cell line.

[0032] Further, Blasticidin is used for drug screening.

[0033] Further, the time for Blasticidin drug screening is 3 - 4 days.

[0034] Further, the time for Blasticidin drug screening is 4 days.

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

[0036] (1) Constructing a stable cell line stably expressing an m 6 A 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] Connecting the gRNA described in (2) to a gRNA vector;

[0039] The sequence of the vector is sequentially connected in the manner of DR sequence, SG sequence, and T*n, where n ≥ 7.

[0040] Further, the sequence of the vector is sequentially connected in the manner of DR sequence, SG sequence, and T*7, where the DR sequence is as shown in SEQ ID NO: 2, and the SG sequence is the gRNA sequence.

[0041] Further, in step (1), the m 6 A site-directed erasure tool refers to the fusion of a dCas protein and the catalytic domain of a demethylase.

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

[0043] Further, 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 the 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 the guidance of gRNA.

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

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

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

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

[0049] Further, Blasticidin is used for drug screening.

[0050] Further, the time for Blasticidin drug screening is 3 - 4 days.

[0051] Further, the time for drug screening is 4 days.

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

[0053] Further, the tumor cell line includes colorectal cancer cell line, liver cancer cell line, lung cancer cell line, and gastric cancer cell line.

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

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

[0056] The fifth aspect of the present invention provides a single - cell level m 6 A unit - point CRISPR screening system constructed by the method described in the second aspect of the present invention.

[0057] The sixth aspect of the present invention provides a transcriptome - level m 6 A unit - point CRISPR screening system constructed by the method described in the third aspect of the present invention.

[0058] The seventh aspect of the present invention provides the single - cell level m described in the fifth aspect of the present invention6 A single-site CRISPR screening system or the transcriptome-level m described in the sixth aspect of the present invention 6 Application of a single-site CRISPR screening system in screening for m that plays a key role in colorectal cancer liver metastasis 6 Application in A sites.

[0059] The eighth aspect of the present invention provides an m of the SOX2 gene 6 Application of an inhibitor of the 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 sequence of the nucleic acid is sequentially linked in the manner of X 1 -GGCC-CS2-GGCC-X 2 -SG-T*7.

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

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

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

[0066] The present invention first proposes to conduct large-scale high-throughput screening on the m 6 A sites with significantly increased methylation in colorectal cancer liver metastasis at the transcriptome level, and then search for m 6 A sites that play a key role in colorectal cancer liver metastasis, and clarify the mechanism of colorectal cancer liver metastasis from the perspective of m 6 A single-site, providing a new perspective for tumor prevention, diagnosis, and precision treatment.

[0067] m 6 A single-cell CRISPR screening technology, modify the gRNA used for m 6 A single-base editing for subsequent single-cell sequencing, and then use related algorithms (computer software copyright registration number 2023SR0365699) to analyze the m in each cell 6The level of A methylation change is directly correlated with single-cell gene expression data, and thus, hundreds of different m 6 A methylation editing can be analyzed, and single guide RNAs (sgRNAs) with direct gene expression phenotypes in hundreds to tens of thousands of cells can be detected, greatly expanding the operability, scalability, and resolution of high-throughput functional screening at the transcriptome level. Description of the Drawings

[0068] Figure 1 It is a map of differentially expressed genes in liver metastases of colorectal cancer. Among them, 1A is a map of m 6 A methylation modification and differentially expressed genes after liver metastasis, and 1B is a Venn diagram of differentially expressed genes;

[0069] Figure 2 It is a graph of the m 6 A level of the SOX2 gene after Dox induction;

[0070] Figure 3 It is a graph of the gRNA sequence and conformation in m 6 A single-cell CRISPR screening. Among them, 3A is a graph of the Capture Sequence2 sequence used by the gRNA and its location, and 3B is a graph of 8 gRNA vectors;

[0071] Figure 4 It is a schematic diagram of the truncated X 1 、X 2 sequence of the DR sequence;

[0072] Figure 5 It is a graph of the m 6 A level of the modified gRNA conformation;

[0073] Figure 6 It is a graph of the m 6 A-1398 single-site methylation inhibiting tumor migration of the SOX2 gene. Among them, 6A is a graph of tumor migration, and 6B is a statistical graph of the number of tumor migrations. Detailed Implementation Manner

[0074] The following provides definitions of some terms used in this specification. Unless otherwise specified, all technical and scientific terms used herein generally have the same meaning as commonly understood by those 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 single-site CRISPR screening system, and the method includes the following steps:

[0076] (1) Construct a stable cell line stably expressing an m 6 A site-directed erasure tool;

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

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

[0079] In the present invention, the method further includes, after the lentivirus in (2) infects the cell line constructed in (1), subsequently enriching the gRNA through 3 - 4 days of Blasticidin drug screening. After drug screening, first collect the control group cells, and then the remaining cells are those containing gRNA. Then continue to culture the cells for at least 10 days, collect the cells as the experimental group, and then directly perform single - cell transcriptome sequencing using the existing 10x Genomics. Then, combined with the algorithm developed by the patent cooperation applicant (Computer Software Copyright Registration No. 2023SR0365699), directly correlate the single - cell gene expression data and the m 6 A methylation change level, so that hundreds of different m 6 A site methylation editing can be analyzed without the need to know the cell type or markers.

[0080] In the present invention, CRISPR or the CRISPR system is collectively referred to as 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 the active part of tracrRNA), tracr - paired sequences (covering direct repeats and parts of direct repeats involved in tracrRNA processing in the context of the endogenous CRISPR system), guide sequences (also called spacer sequences, gRNA in the context of the endogenous CRISPR system), or other sequences and transcripts from the CRISPR locus.

[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 a particular organism containing endogenous CRISPR, such as Streptococcus pyogenes. Generally, CRISPR is characterized by elements that facilitate the formation of a CRISPR complex at a target sequence site (also referred to as a protospacer in the context of an endogenous CRISPR system). In the context of forming a CRISPR complex, a target sequence is a nucleic acid sequence to which the guide sequence is designed to have complementarity, wherein hybridization between the target sequence and the guide sequence facilitates the formation of the CRISPR complex. Complete complementarity is not necessarily required, as long as there is sufficient complementarity to cause hybridization and facilitate the formation of the CRISPR complex. The target sequence can comprise any polynucleotide, such as a DNA or RNA polynucleotide.

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

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

[0084] The dCas protein includes the dCas13 protein.

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

[0086] Furthermore, the dCas13 protein is selected from the 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 a target RNA under the guidance of a gRNA.

[0087] In some embodiments, the Cas 13a protein is or is derived from a species of the following: 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), Paludibacter propionicigenes (such as Pp WB4), Listeria weihenstephanensis (such as Lw FSL R9-0317), Listeriaceae bacterium (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 879 str. F0557, Chloroflexus aggregans, Demequina aurantiaca, Thalassospira sp. TSL5-1, Pseudobutyrivibrio sp. OR37, Butyrivibrio sp. YAB3001, Leptotrichia sp. Marseille-P3007, Bacteroides ihuae, Porphyromonadaceae bacterium (such as PbKH3CP3RA), Listeria riparia or Insolitispirillum peregrinum.

[0088] In the present invention, m 6 A or m6A can also be referred to as N 6 -methyladenine or N6-methyladenosine.

[0089] In the present invention, the demethylase refers to m 6 A demethylase, 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 hyperplasia and abnormal differentiation of body cells under the action of various initiating and promoting factors. The growth of a tumor is not regulated by the normal physiological regulation of the body, but destroys normal tissues and organs.

[0093] In the present invention, tumors include, but are not limited to, adrenocortical carcinoma, carcinoma of unknown primary site in adults, adult malignant mesothelioma, AIDS-related cancers, anal cancer, appendiceal cancer, astrocytoma (cerebellar astrocytoma, cerebral astrocytoma, childhood cerebral astrocytoma, pineal astrocytoma), basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone tumors, brain cancer, breast cancer, bronchial adenoma / carcinoid, carcinoid tumor, carcinoma of unknown primary, cervical cancer, chronic myeloproliferative disorders, colon cancer, desmoplastic small round cell tumor, endometrial cancer, ependymoma, epitheloid hemangioendothelioma (EHE), esophageal cancer, Ewing tumor sarcoma family, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, gallbladder cancer, gastrointestinal stromal tumor (GIST), gestational trophoblastic tumor, glioma, colorectal cancer, head and neck cancer, heart cancer, liver cancer, islet cell carcinoma (endocrine pancreas), Kaposi sarcoma, kidney cancer (renal cell carcinoma), laryngeal cancer, leukemia (acute lymphoblastic leukemia, chronic lymphocytic leukemia (also known as chronic lymphoid leukemia), chronic myelogenous leukemia (also known as chronic myeloid leukemia), hairy cell leukemia), lip cancer, liposarcoma, lung cancer (non-small cell lung cancer, small cell 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 / osteosarcoma of bone, medulloblastoma, melanoma, Merkel cell carcinoma, occult primary metastatic squamous carcinoma of the neck, multiple endocrine neoplasia syndrome, myeloma, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myxoma, nasal and paranasal sinus cancer, neuroblastoma, ovarian cancer, ovarian epithelial cancer (surface epithelial stromal tumors), ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, parathyroid carcinoma, penile cancer, pharyngeal cancer (hypopharyngeal 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, stomach cancer, testicular cancer, thymoma, thyroid cancer, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer.

[0094] In a preferred embodiment of the present invention, the tumors include colorectal cancer, liver cancer, lung cancer, and stomach cancer.

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

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

[0097] (1) Construct a stable cell line expressing m 6 A stable cell line of a site-directed erasure tool;

[0098] (2) Construct a gRNA library targeting key genes, package it into lentivirus, and infect the cell line in (1);

[0099] The sequence of the vector is sequentially connected in the manner of DR sequence, SG sequence, and T*n, where n≥7.

[0100] Furthermore, the sequence of the vector is sequentially connected in the manner of DR sequence, SG sequence, and T*n, where n is 7.

[0101] In the present invention, the method further includes, after infecting the cell line in (1), enriching gRNA by Blasticidin drug screening for 3-4 days. The cells remaining after drug screening are the cells containing gRNA. First, collect the control group cells, and then treat them with oxaliplatin, a drug for treating colorectal cancer, for screening. Compare the surviving cells and perform high-throughput sequencing (HighThroughputSequencing), also known as next-generation sequencing (Next Generation Sequencing, NGS). If more cells survive than the control group, check which genes are enriched after demethylation and are resistant to oxaliplatin; if fewer cells survive than the control, then the targets after demethylation help oxaliplatin exert its efficacy. Through a colorectal cancer liver metastasis model, establish a transcriptome-level m 6 A methylation high-throughput screening system.

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

[0103] In the present invention, the inhibitor of the m 6 A-1398 single-site methylation level of the SOX2 gene includes small molecule compounds, nucleic acids, and bioactive peptides that can inhibit the m 6 A-1398 single-site methylation level of the SOX2 gene.

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

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

[0106] In the present invention, the pharmaceutically acceptable carrier may comprise inert ingredients which do not unduly inhibit the biological activity of the compound. The pharmaceutically acceptable carrier should be biocompatible, e.g., non-toxic, non-inflammatory, non-immunogenic or without other undesirable reactions or side effects when administered to a subject. Standard pharmaceutical formulation 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), buffering substances (such as tween 80, phosphate, glycine, sorbic acid or potassium sorbate), mixtures of partial glycerides 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, polyvinylpyrrolidone, polyacrylates, waxes, polyethylene-polypropylene oxide-block polymers, methylcellulose, hydroxypropylmethylcellulose, 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; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol, and phosphate buffer solutions, and other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, and, in the judgment of the formulator, coloring agents, release agents, coating agents, sweetening agents, flavoring agents and fragrances, preservatives and antioxidants may also be present in the composition.

[0108] Any orally 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, common carriers include lactose and corn starch. Lubricants such as magnesium stearate are typically also added. For oral administration in the form of capsules, diluents that can be used include lactose and dry corn starch. When an aqueous suspension is required for oral administration, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening agents, flavoring agents or coloring agents can 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 forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents, and emulsifying agents such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 - butanediol, dimethylformamide, oils (especially cottonseed, peanut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols, and fatty acid esters of sorbitan, and mixtures thereof. In addition to the inert diluent, the oral compositions may further include adjuvants such as wetting agents, emulsifying agents, and suspending agents, sweetening agents, flavoring agents, and perfuming agents.

[0110] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is admixed with at least one inert pharmaceutically acceptable excipient or carrier such as sodium citrate or calcium 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 acacia, c) humectants, such as glycerol, d) disintegrating agents, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarders, such as paraffin, f) absorption accelerators, such as quaternary ammonium compounds, g) wetting agents, such as cetyl alcohol and glycerol 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 forms may also contain buffering agents.

[0111] Solid compositions of a similar type may also be employed as fillers in soft and hard gelatin capsules, using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols. 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 art of pharmaceutical formulation. They may optionally contain opacifying agents and may also have a composition such that they release the active ingredient only or preferentially in a certain part of the intestine, optionally in a delayed manner. Examples of embedding compositions that may be used include polymers and waxes. Solid compositions of a similar type may also be employed as fillers in soft and hard gelatin capsules, using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols.

[0112] The microencapsulated forms with one or more of the above excipients can also be used in the present invention. Solid dosage forms such as tablets, troches, 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 pharmaceutical formulation field. 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 other than inert diluents, such as tableting lubricants and other tableting aids such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets, and pills, the dosage forms can also contain buffering agents. They can optionally contain opacifying agents and can also have such a composition that they release the active ingredient only or preferentially in a certain part of the intestine, optionally in a delayed manner. Examples of embedding compositions that can be used include polymers and waxes.

[0113] The present invention will be further illustrated below 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 present invention. Without departing from the scope of the present invention, the main features of the present invention can be used in various embodiments.

[0114] Example 1 Transcriptome-level m 6 A single-site CRISPR screening system

[0115] 1 Experimental materials

[0116] 1.1 Cell culture

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

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

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

[0120] (4) Serum-free 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 lysate: 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] 1.4 SELECT detection reagent

[0139] (20)CutSmart (10×) (B7204S, NEB)

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

[0141] (22) Ligase (M0375S, NEB)

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

[0143] 2 Experimental Methods and Results

[0144] 2.1 Construction of a Stable Cell Line Expressing the m 6 A Site-Specific Erasure Tool in the Colorectal Cancer HCT116 Cell Line

[0145] 1) Digest the well-growing colorectal cancer cell line HCT116 (Wuhan Punosai Life Science Co., Ltd., CL-0096) into single cells with 0.25% Trypsin-EDTA (thermofisher, 25200072), and resuspend and count with serum-reduced medium Opti-MEM TM (thermofisher, 31985070).

[0146] 2) Use the CUY21 EDIT II electroporator from BEX. Add 1 μg of the m 6 A site-specific erasure tool plasmid (or negative control plasmid) and 100 ng of the transposase plasmid to every 1 million HCT116 cells for electroporation;

[0147] 3) Seed the electroporated HCT116 cells in a six-well plate and culture them in McCoy 5A medium for 2 days. Subsequently, change the medium to McCoy5A medium containing Doxycycline hyclate (Dox) and puromycin for drug screening for 7 - 14 days until positive cell clone clusters appear.

[0148] 2.2 Verification of the Expression of the m 6 A Site-Specific Erasure Tool in the Colorectal Cancer Cell Line by Immunofluorescence and Western Blot Methods

[0149] 1) Immunofluorescence: Fixing cell samples: Passage the positive cells after drug screening at a ratio of 1:2. After they adhere to the wall for 24 hours, do not add Dox induction to one well and add Dox induction to the other well. After 48 hours, fix the cells. Fix the cells with 4% paraformaldehyde at room temperature for 15 minutes and wash them three times with PBS; Membrane permeabilization: Permeabilize the membrane with a PBS dilution containing 0.3% Triton-X 100 at room temperature for 30 minutes and wash with PBS solution three times; Blocking: Block with 3% BSA blocking solution for 1 hour, then add the primary antibody and incubate overnight at 4°C or incubate for 2 hours at room temperature; Incubating the secondary antibody: Wash three times with PBS solution, add the fluorescent secondary antibody, and incubate for 1 hour at room temperature in the dark. Statistical analysis of experimental results: Wash three times with PBS solution, incubate with DAPI at room temperature in the dark for 5 minutes, and then observe and take pictures with a laser confocal microscope or a fluorescence microscope after washing with PBS solution.

[0150] 2) Western blot: Protein sample preparation: Passage the positive cells after drug screening at a ratio of 1:2. After they adhere to the wall for 24 hours, do not add Dox induction to one well and add Dox induction to the other well. After another 48 hours, collect the cells, add an appropriate amount of protein lysate, place on ice for 30 minutes for sufficient lysis, centrifuge at 12,000 rpm / min at 4°C for 2 minutes, transfer the supernatant to a new EP tube, and place on ice; Protein quantification: Quantify the extracted protein samples using a BCA kit, calculate the volume required for 30 μg of total protein in the sample, with a final volume of 30 μL, add 5 μL of 6× loading Buffer, and calculate the addition of ddH 2For the O volume, first add water and loading buffer, then add the protein. After pipetting and mixing evenly, boil it in a metal bath at 95°C for 10 min and place it on ice for later use; Prepare the SDS-PAGE gel: Prepare SDS-PAGE gels with a separation gel concentration of 8% and a stacking gel concentration of 5% respectively. Insert the sample well comb and wait for it to solidify; Loading: Take out the prepared SDS-PAGE gel, assemble the electrophoresis tank, remove the sample well comb, add 1×SDS-PAGE electrophoresis buffer, load 30 μg of protein into each well, and add 8 μL of marker to each of the two wells at both ends. To avoid edge effects, it is best to use the wells in the middle for injection; Electrophoresis: Electrophorese at a constant voltage of 80 V until the sample reaches the bottom of the stacking gel and forms a straight line, then adjust the voltage to 120 V for separation; 30 min before the end of electrophoresis, immerse the PVDF membrane in methanol for 15 s for activation. The membrane changes from opaque to semi-transparent without white spots, and then immerse the membrane completely in the transfer buffer for more than 5 min to balance. At the same time, immerse the sponge and filter paper in the transfer buffer; Transfer: After electrophoresis, soak the gel, sponge, filter paper and PVDF membrane in the buffer, prepare a "sponge-filter paper-gel-membrane-filter paper-sponge" sandwich and try to remove the air bubbles as much as possible. Put the sandwich into the transfer device, bury the device in ice, and transfer at a constant current of 200-250 mA for 60-90 min; Blocking: Take out the PVDF membrane after transfer, slightly wash it in 1×TBST, immerse it in the blocking solution, and block it at room temperature with slow shaking for 1 h; Incubate with primary antibody: Cut the membrane into appropriate sizes according to the molecular weights of the target protein and the internal reference protein, immerse it in the primary antibody incubation solution (diluted with the blocking solution), and place it in a shaker at 4°C with slow shaking overnight; Wash the membrane after primary antibody: After incubating with the primary antibody overnight, wash it three times with 1×TBST with shaking, 5 min each time; Incubate with secondary antibody: Immerse the membrane in the secondary antibody incubation solution diluted with the blocking solution and incubate it at room temperature with slow shaking for 1 h; Wash the membrane after secondary antibody: After incubating for 1 h, wash it three times with 1×TBST with shaking, 5 min each time; Drop the chemiluminescent solution onto the membrane and expose the protein bands in a chemiluminescence imager for analysis.

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

[0152] (1) Collect clinical sample data of two pairs of paired colorectal cancer primary sites and liver metastasis sites from the clinical pathology department for MeRIP-seq and RNA-seq sequencing. After combined analysis, 395 genes with significant differences in m 6 A methylation modification and expression levels were found ([ Figure 1 A).

[0153] (2) Use the public database GSE50760 of colorectal cancer liver metastasis for bioinformatics analysis and find 3162 genes with differential expression (P<0.05, |log 2FC|>1), and perform a Venn analysis with the self-test data. A total of 84 common genes were found ( Figure 1 B). These 84 genes were defined as potential key genes affecting liver metastasis of colorectal cancer.

[0154] 2.4 In the colorectal cancer system, use the SELECT method to verify m 6 A site-directed erasure tool plays a role in single-site demethylation

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

[0156] (2) Design 5 sgRNAs for this site. S1 is located upstream of signal A1398, 33 nt away; S2 and S3 cover signal A1398; S4 and S5 are located downstream of signal A1398, with distances of 3 nt and 203 nt respectively. To evaluate the possibility of crRNA off-targeting, non-targeting crRNA (NT) was used as a negative control. The stable cell lines containing different crRNAs were treated with Dox induction respectively, and the methylation level of the m 6 A-1398 site was detected using the SELECT method. The results showed that only under the targeting of S1 and S3, the amount of SELECT product was significantly increased compared with the control group, with statistical significance, indicating that after Dox induction, the m 6 A level at this site was significantly decreased. The experimental results show that the TRME editor can not only cause a decrease in the methylation level of specific m 6 A sites but also has a very precise editing window, indicating that a single-site demethylation system has been successfully constructed in the colorectal cancer HCT116 cell line and can be used for the subsequent screening work (S1-5 represent sgRNA1-5, Figure 2 , Table 1).

[0157] Table 1 sgRNA (gRNA) sequences

[0158]

[0159]

[0160] 2.5 Construct a gRNA library and package lentivirus

[0161] For the 84 potential key genes affecting liver metastasis of colorectal cancer screened above, screen out the possible m 6The A locus and the gRNA sequences targeting the above locus. The patent applicant submitted the gRNA sequences to Genewiz (Suzhou) Inc. for synthesizing chips, and further constructed them onto the gRNA vectors used previously. To ensure that most cells transfected only one gRNA instead of multiple gRNAs simultaneously, lentiviruses with a high transfection efficiency and an MOI less than or equal to 0.3 needed to be constructed. The packaging of this part of the lentiviruses was completed by Ascenta (Suzhou) Co., Ltd.

[0162] 2.6 gRNA library infection of stably expressing m 6 Stably transfected cell line of the A site-directed erasure tool

[0163] (1) Use the synthesized lentiviruses to infect the stably transfected cell line of the m 6 A site-directed erasure tool. To ensure the transfection efficiency, at least 100 - 200 cells were transfected with the same gRNA.

[0164] (2) Since the gRNA vector carried the Blasticidin resistance gene, after the cells were transfected with gRNA, they were screened with Blasticidin for 4 days. The surviving cells were those transfected with the gRNA library.

[0165] (3) Collect the cells before the start of the drug screening as the control group, and then use oxaliplatin (20 uM), a drug for treating colorectal cancer, for screening. Collect the surviving cells as the experimental group. Extract the genomic DNA from the cells of the control group and the experimental group, and send them to Genewiz (Suzhou) Inc. for NSG sequencing and data analysis.

[0166] Example 2 m at the single-cell level 6 Single-site CRISPR screening system for m

[0167] 1 Experimental materials

[0168] 1.1 Cell culture

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

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

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

[0172] (4) Serum-free 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] 1.3 SELECT detection reagent

[0179] (9)CutSmart(10×)(B7204S,NEB)

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

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

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

[0183] 1.4 Transwell experiment

[0184] (13) Polycarbonate membrane cell culture insert(Chamber)(CLS3422,Sigma)

[0185] 2 Experimental methods and results

[0186] 2.1 In the colorectal cancer HCT116 cell line, a stable cell line expressing m 6 A site-directed erasure tool was constructed (the method is the same as in Example 1)

[0187] 2.2 In the colorectal cancer cell line, the expression of m 6 A site-directed erasure tool was verified by immunofluorescence and Western blot methods (the method is the same as in Example 1)

[0188] 2.3 Modify the gRNA vector to make it suitable for single-cell sequencing

[0189] (1) The patent applicant based on the CaptureSequence2 sequence and its location used in the gRNA currently used in single-cell CRISPR screening(Figure 3 A), eight conformations (gRNA vectors, Figure 3 B, Table 2) were designed respectively, and the gRNA vectors for single-site demethylation were modified by homologous recombination. According to the sequencing results, the modification was successful. Among them, the X 1 sequence or the X 2 sequence is a truncated sequence of the DR sequence, and its truncated form is as Figure 4 shown.

[0190] Table 2 Sequences of eight gRNA vectors

[0191]

[0192]

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

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

[0195] (2) Using the SELECT method for detection, as Figure 5 shown, it was found that only under the targeting of the gRNA conformations numbered 4 and 6, the amount of SELECT product increased significantly compared with the control group, and the m 6 A level at this site decreased significantly, showing a statistical difference, indicating that these two gRNA conformations after modification still retained the ability to demethylate a single site.

[0196] (3) The stable cell lines with the above two conformations were respectively subjected to transwell experiments, as Figure 6 shown, and it was found that only under the conformation numbered 6, tumor migration was inhibited. Moreover, the patent applicant first confirmed that the reduction of the m 6 A-1398 single-site methylation level of the SOX2 gene could inhibit tumor migration, so the modified gRNA with this number was selected for subsequent single-cell m 6 A single-site screening.

[0197] 2.4 Identify potential genes affecting the occurrence and development of colorectal cancer and the corresponding m 6 A sites

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

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

[0200] (1) Design gRNAs according to the cas13design: A flexible tool to design Cas13d guide RNAs website (https: / / cas13design.nygenome.org). Design 3 gRNAs for each m 6 A site, then send the gRNA sequences to Suzhou Genewiz Biotechnology Co., Ltd. for chip synthesis, construct them onto the gRNA vector numbered 6 to form a gRNA library, and package it into lentivirus. Similarly, to ensure that most cells only take up one gRNA instead of multiple gRNAs simultaneously, lentivirus with a high transfection efficiency and an MOI less than or equal to 0.3 needs to be constructed. The patent applicant entrusts the packaging of lentivirus to Suzhou Anshengda Company.

[0201] 2.6 Infection of the gRNA library into the stable cell line expressing the m 6 A site-directed erasure tool

[0202] (1) After synthesizing the lentivirus, according to Table 3, infect the stable cell line expressing the m 6 A site-directed erasure tool. To ensure the transfection efficiency, at least 100 - 200 cells are transfected with the same gRNA.

[0203] Table 3 Infection of the stable cell line

[0204]

[0205]

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

[0207] (3) After drug screening, first collect the control group cells, and then the remaining cells are those containing gRNA. Then continue to culture the cells for at least 10 days, collect the cells as the experimental group, send them to the company for single-cell transcriptome sequencing directly using the existing 10x Genomics, and then combine with the algorithm developed by the patent co-applicant (Computer Software Copyright Registration No. 2023SR0365699) to analyze the single-cell gene expression data and the m within the single cell 6It is directly correlated with the level of methylation change.

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

Claims

1. A gRNA vector for single-cell CRISPR screening for m 6 ​ It is characterized in that The gRNA vector is sequentially ligated in the manner of X 1 -GGCC-CS2-GGCC-X 2 -SG-T*7; Among them, the CS2 sequence is as shown in SEQ ID NO: 1, the SG sequence is the gRNA sequence, and the X 1 sequence is as shown in SEQ ID NO: 6, and the X 2 sequence is as shown in SEQ ID NO:

7.

2. A method for constructing a single-cell level m 6 A unit-point CRISPR screening system It is characterized in that The method comprises the following steps: (1) Construct a stable cell line expressing m 6 A stable cell line of the A site-directed erasure tool; (2)According to the m of target shooting 6 Construct a gRNA library based on the A site, package lentivirus, and infect the cell line constructed in (1); The gRNA described in (2) is ligated to the gRNA vector described in claim 1.

3. According to the method described in claim 2, It is characterized in that The MOI of lentiviral infection is less than or equal to 0.

3.

4. According to the method described in claim 2, It is characterized in that When lentiviruses infect cells, at least 200 cells are infected with the same kind of gRNA.

5. According to the method described in claim 2, It is characterized in that When lentiviruses infect cells, at least 100 cells are infected with the same kind of gRNA.

6. According to the method described in claim 2, It is characterized in that The cell line is a tumor cell line.

7. According to the method described in claim 6, It is characterized in that The tumor cell line includes colorectal cancer cell line, liver cancer cell line, lung cancer cell line, gastric cancer cell line.

8. According to the method described in claim 7, It is characterized in that The tumor cell line is selected from colorectal cancer cell lines.

9. According to the method described in claim 2, It is characterized in that In step (1), m 6 A targeted eraser tool refers to the fusion of dCas protein with the catalytic domain of a demethylase.

10. According to the method described in claim 9, It is characterized in that The dCas protein includes dCas13 protein.

11. According to the method described in claim 10, It is characterized in that The dCas13 protein includes dCas13a protein, dCas13b protein, dCas13c protein, dCas13d protein, dCas13X protein, dCas13Y protein.

12. According to the method described in claim 11, It is characterized in that The dCas13 protein is selected from dCas13a protein.

13. According to the method described in claim 9, It is characterized in that The demethylase includes ALKBH5, FTO.

14. According to the method described in claim 13, It is characterized in that The demethylase is selected from ALKBH5.

15. According to the method described in claim 2, It is characterized in that The method further includes the step of drug screening after infecting the cell line.

16. According to the method described in claim 15, It is characterized in that Blasticidin is used for drug screening.

17. According to the method described in claim 16, It is characterized in that The time for Blasticidin drug screening is 3 - 4 days.

18. According to the method described in claim 17, It is characterized in that The time for Blasticidin drug screening is 4 days.

19. Use of the gRNA vector according to claim 1 in m 6 A single-cell CRISPR screening.

20. A single-cell level m constructed by the method according to any one of claims 2-18 6 A single-site CRISPR screening system.

21. The m described in claim 20 at the single-cell level 6 The application of a single-site CRISPR screening system in screening for m that plays a key role in colorectal cancer liver metastasis 6 in site A.

22. Application of inhibitor of single-site methylation level of SOX2 gene m 6 A-1398 in the preparation of a pharmaceutical composition for treating colorectal cancer; The inhibitor is selected from nucleic acids; The sequence of the nucleic acid is X 1 -GGCC-CS2-GGCC-X 2 -SG-T*7 are sequentially linked in this way; Wherein, The CS2 sequence is as shown in SEQ ID NO: 1, X 1 The sequence is as shown in SEQ ID NO: 6, X 2 The sequence is as shown in SEQ ID NO: 7, and the sequence of SG is as shown in SEQ ID NO:

8.

23. According to the application described in claim 22, It is characterized in that The pharmaceutical composition further includes a pharmaceutically acceptable carrier.

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