Controllable RNA m6a demethylation editing system and use thereof

Abstract

Provided is a controllable RNA m6A demethylation editing system, comprising a targeting element and an editing element. The targeting element comprises an inactivated Cas13 protein, and the editing element comprises an FTO and / or an ALKBH5 methyltransferase domain. The editing system can specifically carry out m6A demethylation editing in tumor cells without affecting the m6A demethylation regulation of normal cells; also, no additional light inducer or chemical inducer is needed, thus eliminating side effects of an inducer on cells and organisms.

Description

A controllable RNA m6A demethylation editing system and its application

[0001] This disclosure claims priority to a Chinese patent application filed with the Patent Office of China on September 28, 2023, entitled “A controllable RNA m6A demethylation editing system and its application”, application number 202311278012.6. The entire contents of the above application are incorporated herein by reference. Technical Field

[0002] The present invention belongs to the field of biotechnology and relates to a controllable RNA m6A demethylation editing system and applications thereof. Background Art

[0003] RNA modification is a post-transcriptional regulatory mechanism that is widely present on various RNAs, including messenger RNA, transfer RNA, ribonucleic acid RNA, and non-coding RNA (Qiu L. et al., Mol. Biomed. 2023, 4, 25; Zhang Y. et al., RNA Biol. 2023, 20, 603-613; Barbieri I & Kouzarides T, Nat. Rev. Cancer 2020, 20, 303-322). More than 150 types of RNA modifications have been identified, of which N6-adenine methylation (m6A) is one of the most common RNA modifications and is also the most thoroughly studied type of RNA modification. The RNA m6A regulatory process is divided into three parts: methylases (such as METTL3, METTL14, etc.), demethylases (such as FTO, ALKBH5, etc.), and binding proteins (such as YTHDF1, YTHDF2, etc.). The dynamic changes of m6A and its regulatory proteins have a significant impact on RNA localization, transport, splicing, and transcription, and play a decisive role in intracellular and extracellular signal transduction, cell reprogramming, and cell fate (Zaccara S., et al. Nat. Rev. Mol. Cell Biol. 2019, 20, 608-624; He PC & He C. EMBO J. 2021, 40, e105977). Dysregulation of m6A and its regulatory proteins is closely related to the occurrence and development of various diseases (Wang T., et al. Mol. Cancer 2020, 19, 88; Jiang X., et al. Signal Trans. Target Ther. 2021, 6, 74).

[0004] With the in-depth research and widespread use of CRISPR technology, people have realized that by utilizing the targeting ability of the CRISPR-Cas system and cooperating with the functional domains of m6A methylase or demethylase, a single-base-level m6A methylation and demethylation editing system can be formed. For example, Liu XM et al. used the fusion of Cas9 protein with m6A methylase and demethylase to form a precise editing system (Liu XM et al., Nat. Chem. Biol. 2019, 15, 865-871), while David R. Liu's laboratory further used the Cas13 editing system to improve the specificity of m6A editing and reduce the off-target risk by 5.5 times (Wilson C. et al., Nat. Biotech. 2020, 38, 1431-1440). The above-mentioned first-generation m6A methylation and demethylation editing system cannot freely control the opening and closing of its m6A methylation and demethylation functions. Therefore, when the first-generation m6A methylation and demethylation editing system is added to cells or animals, its m6A methylation and demethylation editing functions will continue to run and cannot be stopped, and there is a possibility of over-editing.

[0005] In the second-generation m6A methylation and demethylation editing system, a switch is designed to control the opening and closing of the m6A methylation and demethylation functions of the system. In most cases, the editing system is in a closed state and does not have the m6A methylation and demethylation functions. However, after adding an inducer (photoinducer or chemical inducer), the editing system can be activated to perform the m6A methylation and demethylation functions. Zhao J et al. assembled the light-sensitive protein with Cas13b, the m6A methylase METTL3-METTL14, and the demethylase FTO, respectively, and controlled the precise editing of m6A by blue light (Zhao J. et al., Small 2020, 16, e1907301). Shi H et al. cut Cas13b into the N-terminal domain and the C-terminal domain, and fused the folate binding proteins PYL and ABI to the two respectively. When folate is bound, Cas13b can be correctly assembled to have editing function (Shi H. et al., Nat. Commun. 2022, 13, 1958). Although the second-generation m6A methylation and demethylation editing system can achieve controllable editing function, the inducers (light or chemical inducers) used therein have a certain effect on cells and can disrupt gene expression and RNA modification status.

[0006] Summary of the Invention

[0007] Problems to be solved by the invention

[0008] Based on the above-mentioned problems existing in the prior art, the purpose of the present invention is to develop a novel controllable m6A methylation and demethylation editing system that does not require additional light inducers or chemical inducers, and has the ability to edit m6A demethylation only in cancer cells without affecting the m6A demethylation regulation of normal cells.

[0009] Solutions for solving problems

[0010] The present invention provides a controllable RNA m6A demethylation editing system, wherein the editing system comprises a targeting element and an editing element;

[0011] Wherein, the targeting element comprises an inactivated Cas13 protein, and the editing element comprises a methyltransferase domain of FTO and / or ALKBH5.

[0012] Preferably, the inactivated Cas13 protein is selected from the full-length or partial sequence of the wild type or mutant of inactivated Cas13a, inactivated Cas13b, inactivated Cas13c, inactivated Cas13d, inactivated Cas13f, inactivated Cas13X and inactivated Cas13Y;

[0013] Preferably, the inactivated Cas13 protein is selected from the full-length or partial sequence of the wild type or mutant of inactivated Cas13a, inactivated Cas13b and inactivated Cas13X;

[0014] Preferably, the inactivated Cas13 protein is selected from the full-length or partial sequence of the wild type or mutant of inactivated Cas13X;

[0015] Preferably, the inactivated Cas13 protein is selected from the full-length or partial sequence of the wild-type or mutant of inactivated Cas13X.1;

[0016] Preferably, the inactivated Cas13 protein comprises one or more of the following sequences:

[0017] (1) the amino acid sequence shown in SEQ ID NO. 1;

[0018] (2) an amino acid sequence that is at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO. 1, and that retains the activity of the amino acid sequence shown in SEQ ID NO. 1;

[0019] (3) an amino acid sequence in which one or more amino acid residues are added, substituted, deleted, or inserted into the amino acid sequence shown in SEQ ID NO. 1, and which retains the activity of the amino acid sequence shown in SEQ ID NO. 1;

[0020] Preferably, the inactivated Cas13 protein comprises the amino acid sequence 198-614 of the wild-type Cas13X.1, more preferably, the inactivated Cas13 protein comprises the amino acid sequence shown in SEQ ID NO.8.

[0021] Preferably, the FTO methyltransferase domain comprises one or more of the following sequences:

[0022] (1) the amino acid sequence shown in SEQ ID NO. 2;

[0023] (2) an amino acid sequence that is at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO. 2, and that retains the activity of the amino acid sequence shown in SEQ ID NO. 2;

[0024] (3) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in SEQ ID NO. 2, and which retains the activity of the amino acid sequence shown in SEQ ID NO. 2.

[0025] Preferably, the methyltransferase domain of ALKBH5 comprises one or more of the following sequences:

[0026] (1) the amino acid sequence shown in SEQ ID NO. 3;

[0027] (2) an amino acid sequence that is at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO. 3, and that retains the activity of the amino acid sequence of SEQ ID NO. 2;

[0028] (3) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in SEQ ID NO. 3, and which retains the activity of the amino acid sequence shown in SEQ ID NO. 3.

[0029] Preferably, the editing element has an active amino acid modified with a compound of formula I,

[0030] Preferably, the active amino acid is lysine and / or tyrosine, more preferably, the active amino acid is tyrosine;

[0031] Preferably, the editing element is an FTO methyltransferase domain in which the active tyrosine residues at positions 106 and 108 in the amino acid sequence are modified with a compound represented by Formula I. More preferably, the amino acid sequence of the modified FTO methyltransferase domain is represented by SEQ ID NO. 4.

[0032] Preferably, the editing element is an ALKBH5 methyltransferase domain in which the active tyrosines at positions 139 and 141 in the amino acid sequence are modified with the compound shown in Formula I. More preferably, the amino acid sequence of the modified ALKBH5 methyltransferase domain is as shown in SEQ ID NO.5.

[0033] The present invention also provides a polynucleotide encoding the methyltransferase domain;

[0034] Preferably, the polynucleotide sequence encoding the FTO methyltransferase domain is as shown in SEQ ID NO.6, or the polynucleotide sequence encoding the FTO methyltransferase domain is complementary to the sequence shown in SEQ ID NO.6;

[0035] Preferably, the polynucleotide sequence encoding the ALKBH5 methyltransferase domain is as shown in SEQ ID NO.7, or the polynucleotide sequence encoding the ALKBH5 methyltransferase domain is complementary to the sequence shown in SEQ ID NO.7.

[0036] The present invention also provides a vector comprising the polynucleotide.

[0037] The present invention also provides a fusion protein, which comprises the targeting element and the editing element;

[0038] Preferably, the fusion protein further comprises a linker, and more preferably, the amino acid sequence of the linker is as shown in SEQ ID NO.9;

[0039] Preferably, the amino acid sequence of the fusion protein is shown in SEQ ID NO.10, or SEQ ID NO.11, or SEQ ID NO.12, or SEQ ID NO.13.

[0040] The present invention also provides a genetically engineered cell, comprising one or more of the editing system, the polynucleotide, the vector and the fusion protein.

[0041] The present invention also provides a use of the editing system, the polynucleotide, the vector, the fusion protein and the genetically engineered cell in the preparation of a drug for regulating RNA demethylation modification.

[0042] Effects of the Invention

[0043] The present invention provides a simple and controllable m6A demethylation editing system that can specifically perform m6A demethylation editing in tumor cells without affecting the m6A demethylation regulation of normal cells. At the same time, the editing system of the present invention does not require additional light-induced or chemical inducers, thereby eliminating the side effects of inducers on cells and the body, and therefore has broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 is a schematic diagram of the m6A demethylation editing efficiency of Cas13x-FTO, Cas13x-ALKBH5, Cas13x-mod-FTO and Cas13x-mod-ALKBH5 in human cervical cancer cell lines (HeLa cells) and human normal cervical epithelial cells (HCerEpiC cells). DETAILED DESCRIPTION

[0045] To make the technical solutions and beneficial effects of the present invention more clearly understood, the following detailed description is given by way of specific embodiments. The accompanying drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly illustrate the details of the local features. Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application belongs.

[0046] The terms and laboratory procedures used in the present invention in connection with protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology are those widely used in the respective fields. For example, standard recombinant DNA and molecular cloning techniques used in the present invention are well known to those skilled in the art and are fully described in Sambrook et al., Molecular Cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, 1989.

[0047] In this specification, a numerical range expressed using "a numerical value A - a numerical value B" means a range including the endpoints A and B.

[0048] As used herein, the term "element" refers to a series of functional nucleic acid sequences useful for protein expression. The "element" described in the present invention is systematically constructed to form an expression construct. The "element" sequence can be the sequence provided in the present invention, but also includes variants thereof, as long as these variants substantially retain the function of the "element".

[0049] As used herein, the terms "nucleic acid" and "nucleic acid molecule" refer to compounds comprising a core base and an acidic moiety, such as a polymer of nucleosides, nucleotides, or nucleotides. Typically, polymeric nucleic acids, such as nucleic acid molecules comprising three or more nucleotides, are linear molecules in which adjacent nucleotides are interconnected by phosphodiester bonds. In certain embodiments, "nucleic acid" refers to a single nucleic acid residue (e.g., nucleotide and / or nucleoside). In certain embodiments, "nucleic acid" refers to an oligonucleotide chain comprising three or more individual nucleotide residues.

[0050] As used herein, the terms "oligonucleotide" and "polynucleotide" are used interchangeably to refer to polymers (e.g., strings of at least three nucleotides) of nucleotides. In certain embodiments, "nucleic acid" includes RNA and single-stranded and / or double-stranded DNA. Nucleic acid can be naturally occurring, such as in the context of a genome, transcript, mRNA, tRNA, rRNA, siRNA, snRNA, plasmid, cosmid, chromosome, chromatid or other naturally occurring nucleic acid molecules. On the other hand, nucleic acid molecules can be non-naturally occurring molecules, such as recombinant DNA or RNA, artificial chromosomes, engineered genomes or their fragments, or synthetic DNA, RNA, DNA / RNA hybrids or include non-naturally occurring nucleotides or nucleosides. In addition, the terms "nucleic acid," "DNA," "RNA" and / or similar terms include nucleic acid analogs, such as analogs with other skeletons except the phosphodiester backbone. Nucleic acid can be purified from natural sources, produced using a recombinant expression system and optionally purified, chemically synthesized, etc. In suitable cases, such as in the case of chemically synthesized molecules, nucleic acid can include nucleoside analogs, such as analogs or sugar and backbone modifications with chemically modified bases.

[0051] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein and refer to amino acid polymers of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component).

[0052] As used herein, the term "fusion protein" refers to a hybrid polypeptide comprising a protein domain from at least two different proteins. A protein can be located at the amino-terminal (N-terminal, N-terminal) portion or carboxyl-terminal (C-terminal, C-terminal) protein of the fusion protein, thereby forming an "amino-terminal fusion protein" or a "carboxyl-terminal fusion protein" respectively. Any protein provided herein can be produced by any method known in the art. For example, provided herein is a protein that can be produced via recombinant protein expression and purification, which is particularly suitable for fusion proteins comprising peptide linkers. Methods for recombinant protein expression and purification are well known, and include those described in: Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)), the entire contents of which are incorporated herein by reference.

[0053] As used herein, the term "Cas protein" refers to CRISPR-associated proteins. Cas proteins and CRISPR sequences together constitute the CRISPR / Cas system. Cas proteins have functional domains related to nucleases and cut target sequences at specific locations by recognizing PAM (protospacer adjacent motif).

[0054] As used herein, the terms “nucleic acid programmable nucleotide binding domain” and “nucleic acid programmable DNA-binding protein (napDNAbp)” refer to proteins that bind to nucleic acids (e.g., DNA or RNA), such as guide polynucleotides (e.g., gRNA), which guide napDNAbp to a specific nucleic acid sequence, for example, by hybridizing with a target nucleic acid sequence. For example, the Cas13 protein can be bound to a guide RNA that guides the Cas9 protein to a specific DNA sequence that is complementary to the guide RNA. In some embodiments, napDNAbp is a Cas13 domain, e.g., nuclease-active Cas13, Cas13 nickase (nCas13) or nuclease-free Cas13 (dCas13). Examples of nucleic acid programmable DNA-binding proteins include, but are not limited to, Cas13 (e.g., dCas13 and nCas13), CasX, CasY, Cpf1, C2c1, C2c2, C2c3, and Argonaute protein (AGO). However, it should be understood that nucleic acid programmable DNA-binding proteins also include nucleic acid programmable proteins that bind RNA. For example, the napDNAbp can be bound to a nucleic acid that guides the napDNAbp to the RNA.Other nucleic acid programmable DNA binding proteins are also within the scope of the present disclosure, although they may not be specifically described in this disclosure.

[0055] As used herein, the term "guide polynucleotide," "guide RNA," or "gRNA" refers to a polynucleotide that can specifically target a target sequence and can form a complex with a nucleic acid programmable nucleotide binding domain protein (e.g., Cas13). In one embodiment, the guide polynucleotide is a guide RNA (gRNA). The gRNA can exist as a complex of two or more RNAs or as a single RNA molecule. A gRNA that exists as a single RNA molecule may be referred to as a single guide RNA (sgRNA), but "gRNA" is used interchangeably to refer to a guide RNA that exists as a single molecule or a complex of two or more molecules. Typically, a gRNA that exists as a single RNA species includes two domains: (1) a domain that has homology to the target nucleic acid (e.g., guiding the binding of the Cas9 complex to the target nucleic acid); (2) a domain that binds to the Cas13 protein. In some embodiments, domain (2) corresponds to a sequence called tracrRNA and includes a stem-loop structure. For example, in some embodiments, domain (2) is identical or homologous to the tracrRNA provided in Jinek et al., Science 337:816-821 (2012). Other examples of gRNAs may be those disclosed in U.S. Provisional Patent Application USSN 61 / 874,682, filed on September 6, 2013, entitled “Switchable Cas9 Nucleases and Uses Thereof” and U.S. Provisional Patent Application USSN 61 / 874,746, filed on September 6, 2013, entitled “Delivery System For Functional Nucleases.” In some embodiments, the gRNA includes two or more of domains (1) and (2) and may be referred to as an “extended gRNA.” The extended gRNA will bind to two or more Cas13 proteins and bind to the target nucleic acid at two or more different regions. The gRNA includes a nucleotide sequence complementary to the target site that mediates the binding of the nuclease / RNA complex to the target site, providing sequence specificity for the nuclease: RNA complex.

[0056] As used herein, the three letter and one letter codes for amino acids are as described in J. biol. chem, 243, p3558 (1968).

[0057] As used herein, amino acid "addition" refers to the addition of amino acids to the C-terminus or N-terminus of an amino acid sequence.

[0058] As used herein, amino acid "deletion" means that 1, 2, or 3 or more amino acids may be deleted from an amino acid sequence.

[0059] As used herein, amino acid "insertion" refers to inserting an amino acid residue into an appropriate position in an amino acid sequence. The inserted amino acid residues may be completely or partially adjacent to each other, or the inserted amino acids may not be adjacent to each other.

[0060] As used herein, amino acid "substitution" refers to the replacement of an amino acid residue at a certain position in an amino acid sequence by another amino acid residue; wherein the "substitution" may be a conservative amino acid substitution.

[0061] As used herein, the term "conservative modification", "conservative substitution" or "conservative replacement" refers to an amino acid in a protein that is replaced by another amino acid with similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation and rigidity, etc.), so that changes can be made frequently without changing the biological activity of the protein. It is known to those skilled in the art that, in general, single amino acid replacements in non-essential regions of a polypeptide do not substantially change the biological activity (see Watson et al. (1987), Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th edition)). In addition, replacements of amino acids with similar structure or function are unlikely to destroy biological activity. Exemplary conservative replacements are set forth in the table below, "Exemplary amino acid conservative replacements".

[0062] As used herein, the terms "coding sequence" and "protein coding sequence" are used interchangeably to refer to a polynucleotide segment that encodes a protein. This region or sequence has a start codon near the 5' end and a stop codon near the 3' end. The coding sequence may also be referred to as an open reading frame.

[0063] As used herein, the terms "complementary" or "hybridize" are used to refer to "polynucleotides" and "oligonucleotides" (which are interchangeable terms referring to nucleotide sequences) in relation to the base pairing rules. For example, the sequence "CAGT" is complementary to the sequence "GTCA". Complementarity can be "partial" or "complete". "Partial" complementarity refers to one or more nucleic acid bases that are mismatched according to the base pairing rules, and "complete" or "complete" complementarity between nucleic acids refers to each nucleic acid base matching another base under the base pairing rules. The degree of complementarity between nucleic acid chains has an important influence on the efficiency and strength of hybridization between nucleic acid chains. This is particularly important in amplification reactions and detection methods that rely on binding between nucleic acids.

[0064] As used herein, the terms "nucleic acid sequence" and "nucleotide sequence" refer to oligonucleotides or polynucleotides and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin that can be single-stranded or double-stranded, and represent the sense or antisense strand.

[0065] As used herein, the terms "sequence identity" and "percentage identity" refer to the percentage of nucleotides or amino acids that are identical (i.e., identical) between two or more polynucleotides or polypeptides. The sequence identity between two or more polynucleotides or polypeptides can be determined by the following method: the nucleotide or amino acid sequences of the polynucleotides or polypeptides are aligned and the number of positions containing the same nucleotide or amino acid residue in the aligned polynucleotides or polypeptides is scored and compared with the number of positions containing different nucleotides or amino acid residues in the aligned polynucleotides or polypeptides. A polynucleotide can differ at one position, for example, by containing different nucleotides (i.e., substitutions or mutations) or missing nucleotides (i.e., nucleotide insertions or nucleotide deletions in one or two polynucleotides). A polypeptide can differ at one position, for example, by containing different amino acids (i.e., substitutions or mutations) or missing amino acids (i.e., amino acid insertions or amino acid deletions in one or two polypeptides). Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of amino acid residues in the polynucleotide or polypeptide. For example, percent identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of nucleotide or amino acid residues in the polynucleotide or polypeptide and multiplying by 100. Exemplary, two or more sequences or subsequences have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleotide "sequence identity" or "percent identity" when compared and aligned for maximum correspondence using a sequence comparison algorithm or as measured by visual inspection. In certain embodiments, the sequences are substantially identical over the entire length of either or both compared biopolymers (e.g., polynucleotides).

[0066] As used herein, the term "vector" refers to a means for introducing a nucleic acid sequence into a cell to produce a transformed cell. Vectors include plasmids, transposons, phages, viruses, liposomes, and episomes. An "expression vector" is a nucleic acid sequence comprising a nucleotide sequence to be expressed in a recipient cell. An expression vector may include additional nucleic acid sequences to facilitate and / or promote expression of the introduced sequence, such as initiation, termination, enhancer, promoter, and secretion sequences.

[0067] As used herein, the terms "individual" and "subject" are used interchangeably and refer to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual is a human.

[0068] The methods disclosed herein can be performed in vitro, ex vivo, or in vivo, or the products can exist in an in vitro, ex vivo, or in vivo form. As used herein, the term "in vitro" refers to experiments using materials, biological substances, cells and / or tissues under laboratory conditions or in culture medium. As used herein, the term "in vivo" refers to experiments and procedures using intact multicellular organisms. In certain embodiments, the methods performed in vivo can be performed on non-human animals. As used herein, the term "ex vivo" refers to an event that exists or occurs outside an organism, such as outside a human or animal, such as an event that can exist or occur on a tissue (e.g., a whole organ) or cell taken from an organism.

[0069] As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., a lubricant, talc, magnesium stearate, calcium or zinc or stearic acid), or solvent encapsulating material, involved in transporting or transporting a compound from one part of the body (e.g., a delivery site) to another part (e.g., an organ, tissue, or part of the body). A pharmaceutically acceptable carrier is "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the tissues of the subject (e.g., physiologically compatible, sterile, physiological pH, etc.). Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants, such as magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, and treacle oil. oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol, and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) pH buffered solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) serum components, such as serum albumin, high-density lipoprotein (HDL), and low-density lipoprotein (LDL); (24) C2-C12 alcohols, such as ethanol; and (25) other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweeteners, flavoring agents, fragrances, preservatives, and antioxidants may also be present in the formulation.Terms such as "excipient," "pharmaceutically acceptable carrier," and the like are used interchangeably herein.

[0070] As used herein, the terms "treatment", "processing" refer to a clinical intervention intended to reverse, alleviate a disease or disorder or one or more symptoms thereof, delay the onset of a disease or disorder or one or more symptoms thereof, or suppress the progression of a disease or disorder or one or more symptoms thereof as described herein. As used herein, the terms "treatment", "processing" refer to a clinical intervention intended to reverse, alleviate a disease or disorder or one or more symptoms thereof, delay the onset of a disease or disorder or one or more symptoms thereof, or suppress the progression of a disease or disorder or one or more symptoms thereof as described herein. In some embodiments, treatment can be administered after one or more symptoms have been formed and / or after the disease has been diagnosed. In other embodiments, treatment can be administered in the absence of symptoms, for example, for preventing or delaying the onset of symptoms or suppressing the onset or progression of a disease. For example, treatment can be administered to susceptible individuals before the onset of symptoms (for example, in view of the history of symptoms and / or in view of genetic or other susceptibility factors). Treatment can also be continued after the symptoms subside, for example to prevent or delay their recurrence.

[0071] The present invention provides a controllable RNA m6A demethylation editing system, wherein the editing system comprises a targeting element and an editing element;

[0072] Wherein, the targeting element comprises an inactivated Cas13 protein, and the editing element comprises a methyltransferase domain of FTO and / or ALKBH5.

[0073] In certain embodiments, the inactivated Cas13 protein is selected from the full-length or partial sequence of a wild type or mutant of inactivated Cas13a, inactivated Cas13b, inactivated Cas13c, inactivated Cas13d, inactivated Cas13f, inactivated Cas13X and inactivated Cas13Y.

[0074] In certain embodiments, the inactivated Cas13 protein is selected from the full-length or partial sequence of a wild-type or mutant of inactivated Cas13a, inactivated Cas13b, and inactivated Cas13X.

[0075] In certain embodiments, the inactivated Cas13 protein is selected from the full-length or partial sequence of a wild-type or mutant of inactivated Cas13X.

[0076] In certain embodiments, the inactivated Cas13 protein is selected from the full-length or partial sequence of the wild-type or mutant of inactivated Cas13X.1.

[0077] In certain embodiments, the inactivated Cas13 protein comprises one or more of the following sequences:

[0078] (1) the amino acid sequence shown in SEQ ID NO. 1;

[0079] (2) an amino acid sequence that is at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO. 1, and that retains the activity of the amino acid sequence shown in SEQ ID NO. 1;

[0080] (3) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in SEQ ID NO.1, and which retains the activity of the amino acid sequence shown in SEQ ID NO.1.

[0081] In certain embodiments, the inactivated Cas13 protein comprises the amino acid sequence shown in SEQ ID NO.1.

[0082] In certain embodiments, the inactivated Cas13 protein comprises an amino acid sequence that is at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO.1, and retains the activity of the amino acid sequence shown in SEQ ID NO.1.

[0083] In certain embodiments, the inactivated Cas13 protein comprises an amino acid sequence in which one or more amino acid residues are added, substituted, deleted, or inserted into the amino acid sequence shown in SEQ ID NO.1, and it retains the activity of the amino acid sequence shown in SEQ ID NO.1.

[0084] In certain embodiments, the inactivated Cas13 protein comprises the amino acid sequence 198-614 of wild-type Cas13X.1.

[0085] In certain embodiments, the inactivated Cas13 protein comprises the amino acid sequence shown in SEQ ID NO.8.

[0086] In certain embodiments, the FTO methyltransferase domain comprises one or more of the following sequences:

[0087] (1) the amino acid sequence shown in SEQ ID NO. 2;

[0088] (2) an amino acid sequence that is at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO. 2, and that retains the activity of the amino acid sequence shown in SEQ ID NO. 2;

[0089] (3) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in SEQ ID NO. 2, and which retains the activity of the amino acid sequence shown in SEQ ID NO. 2.

[0090] In certain embodiments, the FTO methyltransferase domain comprises the amino acid sequence shown in SEQ ID NO.2.

[0091] In certain embodiments, the FTO methyltransferase domain comprises an amino acid sequence that is at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO.2, and it retains the activity of the amino acid sequence shown in SEQ ID NO.2.

[0092] In certain embodiments, the FTO methyltransferase domain comprises an amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in SEQ ID NO.2, and it retains the activity of the amino acid sequence shown in SEQ ID NO.2.

[0093] In certain embodiments, the methyltransferase domain of ALKBH5 comprises one or more of the following sequences:

[0094] (1) the amino acid sequence shown in SEQ ID NO. 3;

[0095] (2) an amino acid sequence that is at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO. 3, and that retains the activity of the amino acid sequence of SEQ ID NO. 2;

[0096] (3) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in SEQ ID NO. 3, and which retains the activity of the amino acid sequence shown in SEQ ID NO. 3.

[0097] In certain embodiments, the methyltransferase domain of ALKBH5 comprises the amino acid sequence shown in SEQ ID NO.3.

[0098] In certain embodiments, the methyltransferase domain of ALKBH5 comprises an amino acid sequence that is at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO.3, and it retains the activity of the amino acid sequence shown in SEQ ID NO.2.

[0099] In certain embodiments, the methyltransferase domain of ALKBH5 comprises an amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in SEQ ID NO.3, and it retains the activity of the amino acid sequence shown in SEQ ID NO.3.

[0100] In certain embodiments, the editing element has an active amino acid modified with a compound of Formula I,

[0101] In certain embodiments, the active amino acid is lysine and / or tyrosine.

[0102] In certain embodiments, the activated amino acid is tyrosine.

[0103] In certain embodiments, the modifications impose chemical modifications on active amino acid side chains.

[0104] In certain embodiments, in normal cells, the protein or domain where the modification is located does not have the m6A demethylation editing function.

[0105] In certain embodiments, in cancer cells, the protein or domain where the modification is located has m6A demethylation editing function.

[0106] In certain embodiments, the editing element is a FTO methyltransferase domain modified with a compound shown in Formula I.

[0107] In certain embodiments, the editing element is an FTO methyltransferase domain in which the active tyrosine residues at positions 106 and 108 in the amino acid sequence are modified with the compound shown in Formula I.

[0108] In this application, the modified tyrosines are all on the surface of the protein, while the others are wrapped inside and cannot be modified.

[0109] In certain embodiments, the amino acid sequence of the modified FTO methyltransferase domain is shown as SEQ ID NO.4.

[0110] wherein X represents tyrosine modified by the compound shown in formula I.

[0111] In certain embodiments, the FTO methyltransferase domain modified by the compound shown in Formula I does not have the m6A demethylation editing function in normal cells.

[0112] In certain embodiments, the FTO methyltransferase domain modified by the compound shown in Formula I has m6A demethylation editing function in cancer cells.

[0113] In certain embodiments, the editing element is an ALKBH5 methyltransferase domain modified with a compound of Formula I.

[0114] In certain embodiments, the editing element is an ALKBH5 methyltransferase domain in which the active tyrosine residues at positions 139 and 141 in the amino acid sequence are modified with the compound of formula I.

[0115] In this application, the modified tyrosines are all on the surface of the protein, while the others are wrapped inside and cannot be modified.

[0116] In certain embodiments, the amino acid sequence of the modified ALKBH5 methyltransferase domain is shown in SEQ ID NO.5.

[0117] wherein X represents tyrosine modified by the compound shown in formula I.

[0118] In this application, the modified polypeptide sequence of the compound represented by Formula I is based on the literature Miller JC, Silverman SK, England PM, Dougherty AD, Lester HA. Flash decaging of tyrosine sidechains in an ion channel. Neuron 1998, 20(4): 619-624.

[0119] In certain embodiments, the ALKBH5 methyltransferase domain modified by the compound of Formula I does not have the m6A demethylation editing function in normal cells.

[0120] In certain embodiments, the ALKBH5 methyltransferase domain modified with the compound of Formula I has m6A demethylation editing function in cancer cells.

[0121] The present invention also provides a polynucleotide encoding the methyltransferase domain.

[0122] In certain embodiments, the polynucleotide encodes the FTO methyltransferase domain.

[0123] In certain embodiments, the polynucleotide sequence encoding the FTO methyltransferase domain is shown as SEQ ID NO.6.

[0124] In certain embodiments, the polynucleotide sequence encoding the FTO methyltransferase domain is complementary to the sequence shown in SEQ ID NO.6.

[0125] In certain embodiments, the polynucleotide encodes the ALKBH5 methyltransferase domain.

[0126] In certain embodiments, the polynucleotide sequence encoding the ALKBH5 methyltransferase domain is shown as SEQ ID NO.7.

[0127] In certain embodiments, the polynucleotide sequence encoding the ALKBH5 methyltransferase domain is complementary to the sequence shown in SEQ ID NO.7.

[0128] The present invention also provides a vector comprising the polynucleotide.

[0129] The present invention also provides a fusion protein, which includes the targeting element and the editing element.

[0130] In certain embodiments, the fusion protein further comprises a linker.

[0131] In certain embodiments, the amino acid sequence of the linker is shown in SEQ ID NO.9.

[0132] SEQ ID NO.9:

[0133] HGEQGDKKRANDNEGT

[0134] In certain embodiments, the amino acid sequence of the fusion protein is shown as SEQ ID NO.10, or SEQ ID NO.11, or SEQ ID NO.12, or SEQ ID NO.13.

[0135] In certain embodiments, the fusion protein is a Cas13x-FTO fusion protein, and the amino acid sequence is shown in SEQ ID NO.10.

[0136] In certain embodiments, the fusion protein is a Cas13x-ALKBH5 fusion protein, and the amino acid sequence is shown in SEQ ID NO.11.

[0137] In certain embodiments, the fusion protein is a Cas13x-mod-FTO fusion protein, and the amino acid sequence is shown in SEQ ID NO.12, wherein X represents tyrosine modified by the compound shown in Formula I.

[0138] In certain embodiments, the fusion protein is a Cas13x-mod-ALKBH5 fusion protein, and the amino acid sequence is shown in SEQ ID NO.13, wherein X represents tyrosine modified by the compound shown in Formula I.

[0139] The present invention also provides a genetically engineered cell, comprising one or more of the editing system, the polynucleotide, the vector and the fusion protein.

[0140] The present invention also provides a use of the editing system, the polynucleotide, the vector, the fusion protein and the genetically engineered cell in the preparation of a drug for regulating RNA demethylation modification.

[0141] Example 1: Construction of an FTO methyltransferase domain with a control switch

[0142] In this example, the methyltransferase domain is selected from the wild-type human demethylase FTO (Uniprot ID: Q9C0B1, whose amino acid sequence is shown in SEQ ID NO. 2), and the active tyrosines at positions 106 and 108 in its amino acid sequence are modified with compound I to obtain an FTO methyltransferase domain with a control switch (hereinafter referred to as mod-FTO), and the specific method is as follows:

[0143] The FTO gene sequence (as shown in SEQ ID NO. 6) was cloned into the pDUAL-HFF1 vector to obtain the recombinant pDUAL-HFF1-FTO plasmid. The pDUAL-HFF1-FTO plasmid was cut with NotI enzyme, and the linear pDUAL-HFF1-FTO fragment was transformed into fission yeast after reacting at 37°C for 30 minutes. YES medium (5 mg / mL yeast extract, 30 mg / mL glucose, 225 μg / mL adenine, 225 μg / mL uracil, 225 μg / mL L-histidine, 225 μg / mL L-leucine, 225 μg / mL L-lysine and 20 mg / mL agar, pH 7.0) was added and cultured to the mid-logarithmic growth phase. The FTO protein was obtained after transformation using the lithium acetate / PEG / heat shock method, and then the protein was expressed according to the literature method (Miller et al. al. Neuron 1998, 20(4): 619-624.) used compound I to modify the active tyrosine to obtain FTO with a control switch (hereinafter referred to as mod-FTO).

[0144] Compound I: (R)-3-(5-(1-hydroxyethyl)-2-nitrophenoxy)-N,N-dimethylbenzamide ((R)-3-(5-(1-hydroxyethyl)-2-nitrophenoxy)-N,N-dimethylbenzamide) (purchased from Accela ChemBio, Cat. SY253115).

[0145] The amino acid sequence of human wild-type demethylase FTO is shown in SEQ ID NO.2:

[0146] The gene sequence of FTO is shown in SEQ ID NO.6:

[0147] The amino acid sequence of the FTO methyltransferase domain with a control switch is shown in SEQ ID NO.4.

[0148] Example 2: Construction of an ALKBH5 methyltransferase domain with a control switch

[0149] In this example, the methyltransferase domain is selected from the wild-type human demethylase ALKBH5 (Uniprot ID: Q6P6C2, whose amino acid sequence is shown in SEQ ID NO. 3), and the active tyrosines at positions 139 and 141 in its amino acid sequence are modified with Compound I to obtain an ALKBH5 methyltransferase domain with a control switch (hereinafter referred to as mod-ALKBH5), and the specific method is as follows:

[0150] The ALKBH5 gene sequence (as shown in SEQ ID NO. 7) was cloned into the pDUAL-HFF1 vector to obtain the recombinant pDUAL-HFF1-ALKBH5 plasmid. The pDUAL-HFF1-ALKBH5 plasmid was cut with NotI enzyme. After reacting at 37°C for 30 minutes, the linear pDUAL-HFF1-ALKBH5 fragment was transformed into fission yeast and cultured in YES medium (5 mg / mL yeast extract, 30 mg / mL glucose, 225 μg / mL adenine, 225 μg / mL uracil, 225 μg / mL L-histidine, 225 μg / mL L-leucine, 225 μg / mL L-lysine and 20 mg / mL agar, pH 7.0) until the mid-logarithmic growth phase. The ALKBH5 protein was obtained after transformation using the lithium acetate / PEG / heat shock method, and then the protein was expressed according to the literature method (Miller et al. Neuron 1998, 20(4): 619-624.) Compound I was used to modify the active tyrosine to obtain ALKBH5 with a control switch (hereinafter referred to as mod-ALKBH5).

[0151] The amino acid sequence of human wild-type demethylase ALKBH5 is shown in SEQ ID NO.3:

[0152] The gene sequence of ALKBH5 is shown in SEQ ID NO.7:

[0153] The amino acid sequence of the ALKBH5 methyltransferase domain with the control switch is shown in SEQ ID NO.5.

[0154] Example 3: Construction of Cas13x-FTO and Cas13x-ALKBH5 with control switches

[0155] In the present embodiment, Cas13x-FTO and Cas13x-ALKBH5 fusion proteins are composed of three parts: targeting element Cas13x, editing element FTO or ALKBH5 and linker. Wherein the targeting element is selected from the 198-614 amino acid sequence (as shown in SEQ ID NO.8) of the wild-type inactivation Cas13X.1 (Uniprot ID: A0A660UUL5, as shown in SEQ ID NO.1), the editing element is selected from FTO or ALKBH5 (its amino acid sequence is shown in SEQ ID NO.2 and SEQ ID NO.3), and the amino acid sequence of the linker is shown in SEQ ID NO.9. After the active tyrosine in Cas13x-FTO and Cas13x-ALKBH5 fusion proteins is modified with compound I, Cas13x-mod-FTO and Cas13x-mod-ALKBH5 fusion proteins with control switches are obtained respectively.

[0156] The amino acid sequence of Cas13X.1 at positions 198-614 is shown in SEQ ID NO.8:

[0157] The amino acid sequence of the linker is shown in SEQ ID NO.9:

[0158] The amino acid sequence of the Cas13x-FTO fusion protein constructed by the aforementioned method is shown in SEQ ID NO.10:

[0159] The amino acid sequence of the Cas13x-ALKBH5 fusion protein constructed by the aforementioned method is shown in SEQ ID NO.11:

[0160] The amino acid sequence of the Cas13x-mod-FTO fusion protein constructed by the aforementioned method is shown in SEQ ID NO.12, wherein X represents tyrosine modified by compound I:

[0161] The amino acid sequence of the Cas13x-mod-ALKBH5 fusion protein constructed by the aforementioned method is shown in SEQ ID NO. 13, wherein X represents tyrosine modified by compound 1:

[0162] Example 4: Evaluation of m6A demethylation editing ability

[0163] To evaluate the m6A demethylation editing ability of Cas13x-FTO, Cas13x-ALKBH5, Cas13x-mod-FTO, and Cas13x-mod-ALKBH5, adenine 48 (A48) of the CYB5A gene was selected as the editing target. A48 of CYB5A is highly methylated in human cervical cancer cells (HeLa cells) and human normal cervical epithelial cells (HCerEpiC cells) (Lin X. et al., Nat. Commun. 2019, 10, 2065). Cas13x-FTO, Cas13x-ALKBH5, Cas13x-mod-FTO, and Cas13x-mod-ALKBH5, along with sgRNA (whose nucleotide sequence is shown in SEQ ID NO. 14), were transfected into HeLa cells and HCerEpiC cells, and the cells were harvested and RNA extracted after 24 hours of culture. 10 μg of RNA was taken and added with a fragmentation reagent (Thermo Fisher Cat. AM8740) for 10 minutes at 70°C to break the mRNA into 100-150 nt fragments. The RNA fragments were precipitated and recovered by ethanol method. The magnetic beads containing protein A and protein G were washed several times with buffer C (150 nM NaCl, 10 mM Tris-HCl, pH 7.5). 5 μg of m6A antibody (Sigma-Aldrich Cat. MABE1006) was added and incubated at 4°C for 2 hours. After washing with buffer C, the magnetic beads were resuspended and RNA fragments were added. The beads were rotated at 4°C overnight. The RNA fragments enriched on the magnetic beads were eluted, reverse transcribed into cDNA, and detected by qPCR. The forward primer sequences and reverse primer sequences used are shown in SEQ ID NO. 15 and SEQ ID NO. 16, respectively.

[0164] The results are shown in Figure 1. In human cervical cancer cell lines (HeLa cells), Cas13x-FTO, Cas13x-ALKBH5, Cas13x-mod-FTO, and Cas13x-mod-ALKBH5 all showed significant m6A demethylation editing ability for the A48 site of CYB5A. In the above system, the m6A methylation level of the A48 site of CYB5A was approximately 60% of that of the blank control. This shows that in tumor cells, the control switches of Cas13x-mod-FTO and Cas13x-mod-ALKBH5 are in the open state and therefore have m6A demethylation editing ability. However, in normal human cervical epithelial cells (HCerEpiC cells), Cas13x-mod-FTO and Cas13x-mod-ALKBH5 do not have m6A demethylation editing ability, while Cas13x-FTO and Cas13x-ALKBH5 can normally exercise their m6A demethylation editing ability. This indicates that in normal cells, the control switches of Cas13x-mod-FTO and Cas13x-mod-ALKBH5 are in the off state, limiting their m6A demethylation editing ability.

[0165] In summary, the m6A demethylation editing ability of Cas13x-mod-FTO and Cas13x-mod-ALKBH5 is cell-specific and only performs m6A demethylation editing on tumor cells.

[0166] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations of the claims. Various modifications and variations may be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form additional embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments merely illustrate several implementations of the present invention and do not limit the scope of protection of the patent of this invention.

Claims

1. A controllable RNAm6A demethylation editing system, characterized in that: The editing system includes a targeting element and an editing element; Wherein, the targeting element comprises an inactivated Cas13 protein, and the editing element comprises a methyltransferase domain of FTO and / or ALKBH5.

2. The editing system according to claim 1, characterized in that: The inactivated Cas13 protein is selected from the full length or partial sequence of the wild type or mutant of inactivated Cas13a, inactivated Cas13b, inactivated Cas13c, inactivated Cas13d, inactivated Cas13f, inactivated Cas13X and inactivated Cas13Y; Preferably, the inactivated Cas13 protein is selected from the full-length or partial sequence of the wild type or mutant of inactivated Cas13a, inactivated Cas13b and inactivated Cas13X; Preferably, the inactivated Cas13 protein is selected from the full-length or partial sequence of the wild type or mutant of the inactivated Cas13X; Preferably, the inactivated Cas13 protein is selected from the full-length or partial sequence of the wild type or mutant of inactivated Cas13X.1; Preferably, the inactivated Cas13 protein comprises one or more of the following sequences: (1) the amino acid sequence shown in SEQ ID NO.1; (2) an amino acid sequence that is at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO.1, and that retains the activity of the amino acid sequence shown in SEQ ID NO.1; (3) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in SEQ ID NO.1, and which retains the activity of the amino acid sequence shown in SEQ ID NO.1; Preferably, the inactivated Cas13 protein comprises the amino acid sequence at positions 198-614 of the wild-type Cas13X.1, and more preferably, the inactivated Cas13 protein comprises the amino acid sequence shown in SEQ ID NO.

8.

3. The editing system according to claim 1, characterized in that: The FTO methyltransferase domain comprises one or more of the following sequences: (1) the amino acid sequence shown in SEQ ID NO.2; (2) an amino acid sequence that is at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO. 2, and that retains the activity of the amino acid sequence shown in SEQ ID NO. 2; (3) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in SEQ ID NO.2, and which retains the activity of the amino acid sequence shown in SEQ ID NO.

2.

4. The editing system according to claim 1, characterized in that: The methyltransferase domain of ALKBH5 comprises one or more of the following sequences: (1) the amino acid sequence shown in SEQ ID NO.3; (2) an amino acid sequence that is at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO.3, and which retains the activity of the amino acid sequence shown in SEQ ID NO.2; (3) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in SEQ ID NO.3, and which retains the activity of the amino acid sequence shown in SEQ ID NO.

3.

5. The editing system according to claim 1, characterized in that: The editing element has an active amino acid modified by a compound shown in Formula I, Preferably, the active amino acid is lysine and / or tyrosine, more preferably, the active amino acid is tyrosine; Preferably, the editing element is a FTO methyltransferase domain in which the active tyrosines at positions 106 and 108 in the amino acid sequence are modified with the compound shown in formula I. More preferably, the amino acid sequence of the modified FTO methyltransferase domain is shown in SEQ ID NO.4; Preferably, the editing element is an ALKBH5 methyltransferase domain in which the active tyrosines at positions 139 and 141 in the amino acid sequence are modified with the compound shown in formula I. More preferably, the amino acid sequence of the modified ALKBH5 methyltransferase domain is as shown in SEQ ID NO.

5.

6. A polynucleotide, characterized in that The polynucleotide encodes a methyltransferase domain according to any one of claims 1 to 5; Preferably, the polynucleotide sequence encoding the FTO methyltransferase domain is as shown in SEQ ID NO.6, or the polynucleotide sequence encoding the FTO methyltransferase domain is complementary to the sequence shown in SEQ ID NO.6; Preferably, the polynucleotide sequence encoding the ALKBH5 methyltransferase domain is as shown in SEQ ID NO.7, or the polynucleotide sequence encoding the ALKBH5 methyltransferase domain is complementary to the sequence shown in SEQ ID NO.

7.

7. A carrier, characterized in that The vector comprises the polynucleotide according to claim 6.

8. A fusion protein, characterized in that The fusion protein comprises a targeting element and an editing element according to any one of claims 1 to 5; Preferably, the fusion protein further comprises a linker, and more preferably, the amino acid sequence of the linker is as shown in SEQ ID NO.9; Preferably, the amino acid sequence of the fusion protein is shown in SEQ ID NO.10, or SEQ ID NO.11, or SEQ ID NO.12, or SEQ ID NO.

13.

9. A genetically engineered cell, characterized in that: The cell comprises one or more of the editing system according to any one of claims 1 to 5, the polynucleotide according to claim 6, the vector according to claim 7, and the fusion protein according to claim 8.

10. Use of the editing system according to any one of claims 1 to 5, the polynucleotide according to claim 6, the vector according to claim 7, the fusion protein according to claim 8 and the genetically engineered cell according to claim 9 in the preparation of a drug for regulating RNA demethylation modification.

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