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WO2021169925A1 - Protéine de fusion et son utilisation - Google Patents

Protéine de fusion et son utilisation Download PDF

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Publication number
WO2021169925A1
WO2021169925A1 PCT/CN2021/077328 CN2021077328W WO2021169925A1 WO 2021169925 A1 WO2021169925 A1 WO 2021169925A1 CN 2021077328 W CN2021077328 W CN 2021077328W WO 2021169925 A1 WO2021169925 A1 WO 2021169925A1
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fusion protein
nucleic acid
sequence
present
combination
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Chinese (zh)
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牛小牧
李彦莎
梁亚峰
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Shandong Shunfeng Biotechnology Co Ltd
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Shandong Shunfeng Biotechnology Co Ltd
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    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
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    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor

Definitions

  • the invention belongs to the field of biotechnology, and specifically relates to a fusion protein and its application.
  • DNA methylation is a form of DNA chemical modification, which can change the genetic performance without changing the DNA sequence. It is a common modification method in eukaryotic cells. DNA methylation is established by DNA methylation transferase using S-adenosylmethionine (SAM) as a methyl donor to catalyze the reaction.
  • SAM S-adenosylmethionine
  • the base at the modified site can be N-6 of adenine (6mA), N-4 of cytosine, N-7 of guanine (7mG) and cytosine The C-5 position (5mC). They are catalyzed by different DNA methylases. However, the most clear and common research is the methylation of 5mC, the C-5 position of cytosine.
  • Methylated DNA can be demethylated.
  • Passive demethylation is related to half-reserved DNA replication. Because the new strands produced by DNA replication do not have DNA methylation, if the methylation maintenance system does not work, this will lead to the occurrence of DNA demethylation. Obviously, this is a passive process.
  • Active demethylation is related to the catalysis of DNA demethylase. For example, TET1 (ten-eleven translocation 1) and ROS1 (repressor of silence 1) are demethylases of animals and plants, respectively. They cannot directly remove the methyl group at the C-5 position of cytosine. The mechanism of base mismatch repair introduces a new unmodified cytosine.
  • DNA demethylation plays an important role in the reactivation of silent genes.
  • DNA methylation can lead to changes in DNA conformation in certain regions, thereby affecting the interaction between protein and DNA, leading to gene silencing.
  • DNA methylation controls a variety of biological processes, including flower morphology, sex determination, plant structure, flowering time, biomass, and leaf senescence.
  • the epigenetic traits of organisms can be manipulated. Therefore, the development of targeted nucleic acid methylation or demethylation tools has important scientific value for the study of methylation function and epigenetic breeding.
  • the present invention provides a protein capable of demethylating and modifying nucleic acid efficiently and site-specifically and its application.
  • a fusion protein comprising components selected from the following:
  • the positioning function element D1 which has the function of targeting and binding DNA
  • Demethylation functional element D2 which has the function of converting methylated nucleotides into non-methylated nucleotides.
  • the D1 element has no catalytic activity and is selected from the following group: Cas protein, zinc finger protein or TALENs protein, or functional domains thereof, or a combination thereof.
  • the D1 element is selected from the following group: dCas9, dCpf1, dCas12, dCas13, dCms1, dMAD7, or functional domains thereof, or combinations thereof.
  • the D1 element is dCas9.
  • the D1 element comprises a sequence selected from the following, or consists of a sequence selected from the following:
  • sequence shown in SEQ ID NO:1 has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, A sequence with at least 97%, at least 98%, or at least 99% sequence identity.
  • the D2 element has the function of converting methylated cytosine into unmethylated cytosine.
  • the D2 element is a demethylase or its demethylation functional domain selected from the group consisting of ROS1, TET, DME, DML, or a combination thereof.
  • the D2 element is ROS1 or its functional domain.
  • the D2 element comprises a sequence selected from the following, or consists of a sequence selected from the following:
  • SEQ ID NO: 3 has one or more amino acid substitutions, deletions or additions (for example, 1, 2, 3, 4, 5, 6, 7, 8 1, 9, or 10 amino acid substitutions, deletions or additions) sequence; or
  • sequence shown in SEQ ID NO: 3 has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least A sequence of 97%, at least 98%, or at least 99% sequence identity.
  • the D1 element is located at the N end or C end of the D2 element.
  • the D1 element and the D2 element are connected by one or more of the following components: peptide bond, connecting peptide, nuclear localization signal, epitope tag, or a combination thereof.
  • the nuclear localization signal comprises a sequence selected from the following, or consists of a sequence selected from the following:
  • sequence shown in SEQ ID NO: 5 or SEQ ID NO: 7 has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%. %, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • the epitope tag is selected from the group consisting of His tag, GST tag, HA tag, c-Myc tag, Flag tag, V5 tag, or a combination thereof.
  • the fusion protein comprises a sequence selected from the following, or consists of a sequence selected from the following:
  • sequence shown in SEQ ID NO: 9 has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, A sequence with at least 97%, at least 98%, or at least 99% sequence identity.
  • the N-terminal or C-terminal of the fusion protein further includes one or more of the following elements: epitope tag, reporter gene sequence, nuclear localization signal (NLS), chloroplast signal peptide, transcription activation domain (E.g., VP64), transcription repression domain (e.g. KRAB structure and or SID domain), nuclease domain (e.g. Fok1), or a combination thereof.
  • epitope tag reporter gene sequence
  • NLS nuclear localization signal
  • chloroplast signal peptide E.g., VP64
  • transcription repression domain e.g. KRAB structure and or SID domain
  • nuclease domain e.g. Fok1
  • a fusion protein combination in the second aspect of the present invention, includes a first fusion protein and a second fusion protein, and the structure of the first fusion protein and the second fusion protein is the same as the present
  • the fusion protein according to the first aspect of the invention is shown; it is characterized in that D2 in the first fusion protein and the second fusion protein are different;
  • the first fusion protein or the second fusion protein has the structure shown in formula I from the N-terminus to the C-terminus;
  • D1 is a positioning function element, which has the function of targeting and binding DNA
  • D2 is a demethylated functional element, which has the function of converting methylated nucleotides into unmethylated nucleotides
  • X is connecting peptide, epitope tag or nuclear localization signal (NLS);
  • n an integer of 0-6;
  • D2 in the respective structures of the first fusion protein and the second fusion protein are different.
  • n 1
  • X is a nuclear localization signal.
  • D2 of the first fusion protein is ROS1 or its functional domain
  • D2 of the second fusion protein is TET1 or its functional domain
  • nucleic acid encoding the fusion protein according to the first aspect of the present invention.
  • sequence of the nucleic acid includes the following elements:
  • Z1 is the nucleotide sequence encoding the positioning function element D1 in the fusion protein.
  • Z2 is the nucleotide sequence encoding the functional demethylation element D2 in the fusion protein.
  • the Z1 element comprises a sequence selected from the following, or consists of a sequence selected from the following:
  • sequence shown in SEQ ID NO: 2 has at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% Sequence of sequence identity;
  • the Z2 element comprises a sequence selected from the following, or consists of a sequence selected from the following:
  • sequence shown in SEQ ID NO: 4 has at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% Sequence of sequence identity;
  • the nucleic acid comprises a sequence encoding a nuclear localization signal
  • the sequence encoding the nuclear localization signal has a sequence selected from the following, or consists of a sequence selected from the following:
  • sequence shown in any one of SEQ ID NO: 6 or SEQ ID NO: 8 has at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% , A sequence with at least 90%, at least 95% sequence identity;
  • the nucleic acid has a sequence selected from the following, or consists of a sequence selected from the following:
  • sequence shown in SEQ ID NO: 10 has at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% Sequence of sequence identity;
  • nucleic acid construct comprising a first nucleic acid sequence and one or more second nucleic acid sequences, wherein the first nucleic acid sequence encodes the fusion protein according to the first aspect of the present invention Or the fusion protein combination according to the second aspect of the present invention, wherein the second nucleic acid sequence is a gRNA coding sequence.
  • the 5 'and / or 3' of the end of the first nucleic acid sequence comprises one or more nuclear localization signal.
  • one end of the first nucleic acid sequence contains a promoter, and optionally, the other end contains a terminator; the promoter is selected from RNA polymerase II-dependent promoters, and the promoter The sub is selected from UBI, UBQ, 35S, Actin, SPL, CmYLCV, YAO, CDC45, rbcS, rbcL, PsGNS2, UEP1, TobRB7, Cab, or a combination thereof.
  • the nucleic acid construct contains 1-6 gRNA coding sequences.
  • the coding sequence of the gRNA series distribution at the 5 'end or 3' end of the first nucleic acid sequence is a preferred embodiment.
  • the second nucleic acid sequences are distributed at both ends of the first nucleic acid structure sequence.
  • each gRNA coding sequence in the second nucleic acid sequence contains an RNA polymerase III-dependent promoter promoter, and the promoter is selected from: U6, U3, U6a , U6b, U6c, U6-1, U3b, U3d, U6-26, U6-29, 7SL or 5H1.
  • a vector which contains the nucleic acid according to the third aspect of the present invention or the nucleic acid construct according to the fourth aspect of the present invention.
  • a protein component comprising the fusion protein described in the first aspect of the present invention or the fusion protein combination described in the second aspect of the present invention.
  • the nucleic acid component is one or more gRNA sequences
  • the protein component and the nucleic acid component combine with each other to form the complex.
  • a polynucleotide combination is provided, which encodes the fusion protein combination according to the second aspect of the present invention.
  • the polynucleotide combination includes a first polynucleotide and a second polynucleotide, wherein both the first polynucleotide and the second nucleotide encodes as in the present invention
  • the fusion protein described in the first aspect, and the D2 elements of the two fusion proteins are different.
  • first nucleotide and the second nucleotide respectively further include one or more gRNA coding sequences.
  • first polynucleotide and the second nucleotide are located in the same vector or different vectors.
  • first polynucleotide and the second nucleotide are located in different vectors.
  • the vector containing the first nucleic acid and the vector containing the second nucleic acid transform cells simultaneously or sequentially.
  • a host cell containing the fusion protein according to the first aspect of the present invention, or the fusion protein combination according to the second aspect of the present invention, or the first aspect of the present invention.
  • the vector of the fifth aspect, or the complex of the sixth aspect of the present invention, or the polynucleotide of the third aspect of the present invention or the polynucleotide of the fourth aspect of the present invention integrated in the genome of the host cell The nucleic acid construct, or the polynucleotide combination according to the seventh aspect of the present invention.
  • the host cell is a eukaryotic cell or a prokaryotic cell.
  • the host cell is a plant cell.
  • the plant is a monocotyledonous plant or a dicotyledonous plant.
  • the ninth aspect of the present invention provides a method for preparing the fusion protein of the first aspect of the present invention, which includes the following steps:
  • the host cell contains the vector according to the fifth aspect of the present invention, or the polynucleotide according to the third aspect of the present invention is integrated into the genome.
  • the fusion protein according to the first aspect of the present invention, or the fusion protein combination according to the second aspect of the present invention, or the nucleic acid according to the third aspect of the present invention, or the fourth aspect of the present invention is provided.
  • the nucleic acid construct described in the aspect, or the vector described in the fifth aspect of the present invention, or the complex described in the sixth aspect of the present invention, or the polynucleotide combination described in the seventh aspect of the present invention is used to remove the target nucleic acid. Use in methylation modification.
  • the demethylation is the conversion of methylated cytosine to unmethylated cytosine.
  • the target nucleic acid is derived from a eukaryote or a prokaryote.
  • the target nucleic acid is derived from plant cells or animal cells.
  • the target nucleic acid is derived from the nucleus, cytoplasm, chloroplast or mitochondria.
  • the target nucleic acid is DNA, RNA or a combination thereof.
  • the fusion protein according to the first aspect of the present invention, or the fusion protein combination according to the second aspect of the present invention, or the nucleic acid according to the third aspect of the present invention, or the first aspect of the present invention is provided.
  • the nucleic acid construct described in the fourth aspect, or the vector described in the fifth aspect of the present invention, or the complex described in the sixth aspect of the present invention, or the polynucleotide combination described in the seventh aspect of the present invention is prepared for Use in a kit for demethylation modification of target nucleic acid.
  • kits comprising one or more of the following group: the fusion protein according to the first aspect of the present invention, or the fusion protein combination according to the second aspect of the present invention , Or the nucleic acid according to the third aspect of the present invention, or the nucleic acid construct according to the fourth aspect of the present invention, the vector according to the fifth aspect of the present invention, the complex according to the sixth aspect of the present invention, the seventh aspect of the present invention.
  • a method for reducing DNA methylation of a target gene or its promoter or its enhancer in a cell expresses the fusion described in the first aspect of the present invention in the cell.
  • Protein, and one or more gRNAs related to the target gene are provided.
  • the fourteenth aspect of the present invention provides a method for regulating the expression of a target gene, which includes the following steps: expressing the fusion protein described in the first aspect of the present invention and combining it with the target gene or the expression control element of the target gene, Demethylate the DNA at this site.
  • the regulation includes: activation, enhancement, inhibition, reduction or inactivation.
  • the present invention provides a method for activating or enhancing gene expression, which comprises the following steps: expressing the fusion protein described in the first aspect of the present invention and combining it with the expression control element of the target gene, Demethylate the DNA at this site.
  • the expression control elements include: promoter, enhancer, terminator, transposon, and silencer.
  • a method for regulating plant traits is provided, which is characterized by comprising the following steps:
  • nucleic acid sequence expressing the gRNA related to the fusion protein and the regulatory gene of the first aspect of the present invention is introduced into the plant cell and integrated into the genome;
  • the method for introducing cells includes Agrobacterium infection, gene gun transformation, microinjection, electric shock, ultrasound, and polyethylene glycol (PEG)-mediated method.
  • the traits are epigenetic traits of plants.
  • Figure 1 shows the decrease in the methylation level of MEMS in the target region in the transgenic T1 plant in Example 1.
  • Figure 2 shows the expression level of ROS1 in the transgenic T1 plant in Example 1.
  • Figure 3 shows the genetic stability of MEMS site demethylation in the transgenic T2 plant in Example 1.
  • Figure 4 shows the results of demethylation of different regions in Example 1.
  • Figure 5 shows the genetic stability of the transgenic T2 plants in Example 2.
  • Figure 6 shows the structural composition of the demethylated gene editing tool.
  • the inventors developed an efficient and site-specific method for removing DNA methylation modification for the first time. Specifically, the present inventors fused dCas9 or its functional domain with the function of targeting and binding DNA with the demethylase ROS1 or its functional domain to obtain a fusion protein; and introduced multiple nucleic acids corresponding to the target. The sequence of the gRNA sequence can be accurately positioned to demethylate the target nucleic acid region.
  • the demethylation method of the present invention has precise and efficient demethylation modification efficiency in plants, and has important scientific value for studying epigenetics of plants and regulating plant traits through demethylation. On this basis, the present invention has been completed.
  • fusion protein refers to the fusion protein described in the first aspect of the present invention, which has the function of targeted binding to DNA and converting target methylated nucleotides into unmethylated nucleotides.
  • fusion protein combination refers to a combination of multiple fusion proteins in the present invention.
  • each fusion protein has a different demethylase catalytic domain.
  • the different demethylase catalytic domains have different demethylation effects on different target nucleic acid sites, so that they complement each other.
  • Cas protein refers to a nuclease.
  • a preferred Cas protein is the Cas9 protein.
  • Typical Cas9 proteins include (but are not limited to): Cas9 derived from Staphylococcus aureus.
  • the Cas9 protein can also be replaced by Cas proteins derived from other CRISPR systems, such as Cpf1 nuclease.
  • the source of the Cpf1 nuclease is selected from the group consisting of Acidaminococcus and Laureus sp. Family (Lachnospiraceae), acid aminococcus mutants, Lachnospiraceae mutants.
  • the “d” in the "dCas9, dCpf1, dCas12, dCas13, dCms1, dMAD7” stands for "dead”, which means Cas protein that has lost its enzymatic cleavage activity, that is, it cannot cut single-stranded or double-stranded DNA sequences, but can still interact with The gRNA forms a complex that targets and binds to the DNA sequence.
  • epitope tag can be fused to the N-terminus or C-terminus of the target protein through molecular genetics, without affecting the biological activity of the target protein, and it is easy to detect with the target protein. .
  • the "connecting peptide” is a short peptide chain composed of multiple amino acids that connects the D1 element and the D2 element to form a fusion protein.
  • the connecting peptide does not affect the expression of the fusion protein.
  • the length of the connecting peptide is generally 1-100 aa, preferably, 15-85 aa, more preferably, 25-70 aa, more preferably, 24-32 aa.
  • the commonly used connecting peptide can be XTEN.
  • gRNA is also called guide RNA or guide RNA, and has the meaning commonly understood by those skilled in the art.
  • guide RNAs can include direct repeats and guide sequences, or consist essentially of direct repeats and guide sequences (also called spacers in the context of endogenous CRISPR systems). (spacer)) composition.
  • gRNA can include crRNA and tracrRNA, or only crRNA, depending on the Cas protein it depends on.
  • crRNA and tracrRNA can be artificially modified and fused to form single guide RNA (sgRNA).
  • the gRNA of the present invention may be natural, or artificially modified or designed and synthesized.
  • the targeting sequence is any polynucleotide sequence that has sufficient complementarity with the target sequence to hybridize with the target sequence and guide the specific binding of the CRISPR/Cas complex to the target sequence, usually having 17- Sequence length of 23nt.
  • the degree of complementarity between the targeting sequence and its corresponding target sequence is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, Or at least 99%. Determining the best alignment is within the abilities of those of ordinary skill in the art. For example, there are published and commercially available alignment algorithms and programs, such as but not limited to ClustalW, Smith-Waterman algorithm in matlab, Bowtie, Geneious, Biopython, and SeqMan.
  • the "functional domain” refers to a region of a protein or enzyme that independently performs its biological function and has a specific structure. It can be a part of the protein structure, or it can be composed of one or more protein domains in an operably linked manner.
  • the domain is a combination of different secondary structures and super-secondary structures, and is a subunit that bears part or all of the physiological functions in the expression of protein functions.
  • the number of amino acid residues in common domains is between 100 and 400, the smallest domain has only 40 to 50 amino acid residues, and the larger domain can exceed 400 amino acid residues.
  • epigenetic refers to a genetic function that has undergone heritable changes in the absence of changes in the DNA sequence of a gene, which ultimately leads to a change in phenotype.
  • mechanisms affecting epigenetics include the following: DNA modification (such as DNA methylation), protein covalent modification, paramutation, regulation of non-coding RNA, chromatin remodeling, or genome imprinting.
  • the "epigenetic traits” mentioned herein refer to the observable plant traits or characteristics controlled by or involved in the regulation of epigenetic mechanisms in plants.
  • the demethylation modification described in the present invention mainly refers to the modification of 5-methylcytosine (5mC), which is a reversible epigenetic modification and plays an important role in the growth and development of plants.
  • 5mC 5-methylcytosine
  • Common demethylases in plants include but are not limited to: ROS1, TET1, DME, DML, etc.
  • ROS1 is a dual-function glycosidase that can directly excise methylated cytosine to create an empty base site, and then initiate base mismatch repair to introduce an unmodified cytosine.
  • TET is a dioxygenase that can oxidize methylated cytosine to 5-hydroxymethylcytosine, and then further catalyze it to 5-formylcytosine and 5-carboxycytosine, and then pass the DNA sugar group
  • the enzyme (TDG) cuts off 5-formylcytosine or 5-carboxycytosine to create an empty base site, which initiates base mismatch repair and reintroduces an unmodified cytosine.
  • the present invention provides a fusion protein, which has the function of targeted binding to DNA and converting target methylated nucleotides into unmethylated nucleotides.
  • the D1 element has no catalytic activity and is selected from the following group: Cas protein, zinc finger protein or TALENs protein, or functional domains thereof, or a combination thereof.
  • the D1 element is selected from the following group: dCas9, dCpf1, dCas12, dCas13, dCms1, dMAD7, or a combination thereof.
  • the D1 element is dCas9.
  • the D1 element is a functional domain of the dCas9 protein, comprising or consisting of the amino acid sequence shown in SEQ ID NO:1; its corresponding coding nucleotide sequence is as SEQ ID NO: 2 shown.
  • the D2 element has the function of converting methylated cytosine into unmethylated cytosine.
  • the D2 element is a demethylase or its demethylation functional domain selected from the following group: ROS1, TET, DME, DML, or a combination thereof; preferably, the D2 element is ROS1 or its function area.
  • the D2 element is a functional domain of the ROS1 protein, comprising or consisting of the amino acid sequence shown in SEQ ID NO: 3; its corresponding coding nucleotide sequence is shown in SEQ ID NO: 4 Shown.
  • the D1 element and the D2 element are connected by one or more of the following components: peptide bond, connecting peptide, nuclear localization signal, epitope tag, or a combination thereof.
  • the nuclear localization signal comprises or consists of the amino acid sequence shown in SEQ ID NO: 5 or SEQ ID NO: 7; the corresponding coding nucleotide sequences of each thereof are as SEQ ID NO: 6 and SEQ ID NO: Shown at 8.
  • the fusion protein comprises a sequence selected from the following, or consists of a sequence selected from the following:
  • sequence shown in SEQ ID NO: 9 has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, A sequence with at least 97%, at least 98%, or at least 99% sequence identity.
  • the N-terminal or C-terminal of the fusion protein further includes one or more of the following elements: epitope tag, reporter gene sequence, nuclear localization signal (NLS), chloroplast signal peptide, transcription activation A domain (for example, VP64), a transcription repression domain (for example, a KRAB structure and or a SID domain), a nuclease domain (for example, Fok1), or a combination thereof.
  • epitope tag for example, reporter gene sequence, nuclear localization signal (NLS), chloroplast signal peptide, transcription activation A domain (for example, VP64), a transcription repression domain (for example, a KRAB structure and or a SID domain), a nuclease domain (for example, Fok1), or a combination thereof.
  • the present invention also includes fragments and analogs having the functions of the fusion protein of the present invention.
  • fragment and analogs refer to polypeptides that substantially maintain the same biological function or activity as the fusion protein of the present invention.
  • the fusion protein fragment, derivative or analogue of the present invention may be: (i) a polypeptide in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acids
  • the residue may or may not be encoded by the genetic code; or (ii) a polypeptide with substitution groups in one or more amino acid residues; or (iii) the mature polypeptide and another compound (such as a compound that prolongs the half-life of the polypeptide) Such as polyethylene glycol) fused to a polypeptide; or (iv) additional amino acid sequence fused to the polypeptide sequence to form a polypeptide (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, Or fusion protein).
  • these fragments, derivatives and analogs belong to the scope well known to those skilled in the art.
  • the said fusion protein variant is the amino acid sequence shown in SEQ ID NO: 9, after several (usually 1-60, preferably 1-30, more preferably 1- 20, preferably 1-10) derived sequences obtained by substituting, deleting or adding at least one amino acid, and adding one or several (usually within 20, preferably 10) at the C-terminus and/or N-terminus Within 5) amino acids.
  • amino acids with similar or similar properties are substituted, the function of the protein is usually not changed, and the addition of one or several amino acids to the C-terminal and/or ⁇ terminal usually does not change the function of the protein.
  • the present invention also includes analogs of the claimed fusion protein.
  • the difference between these analogs and the sequence SEQ ID NO: 9 of the present invention may be the difference in the amino acid sequence, the difference in the modified form that does not affect the sequence, or both.
  • Analogs of these proteins include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, site-directed mutagenesis or other known biological techniques. Analogs also include analogs having residues different from natural L-amino acids (such as D-amino acids), and analogs having non-naturally occurring or synthetic amino acids (such as ⁇ , ⁇ -amino acids). It should be understood that the protein of the present invention is not limited to the representative proteins exemplified above.
  • Modifications include: chemically derived forms of proteins in vivo or in vitro, the modifications can maintain or enhance or partially inhibit the transport function of the protein; the modifications include chemical modifications of amino acid side chains, peptides
  • the chemical modification of the chain end group such as the chemical modification of the sulfhydryl group, the chemical modification of the amino group, the chemical modification of the carboxyl group, the chemical modification of the disulfide bond and other modifications; the chemical modification includes phosphorylation modification (such as phosphotyrosine, Phosphoserine, phosphothreonine), glycosylation modification (mediated by glycosylase, such as N-glycosylation, O-glycosylation), fatty acylation (such as acetylation, palmitoylation), etc. .
  • the present invention also relates to methods for producing fusion proteins or fragments, derivatives or analogs thereof. It includes (a) culturing the above-mentioned host cell under conditions conducive to the production of the fusion protein or its fragment, derivative or analogue; and (b) isolating the fusion protein or its fragment, derivative or analogue.
  • the cells are cultured on a nutrient medium suitable for the production of the fusion protein by a method well known in the art. If the polypeptide is secreted into the nutrient medium, the polypeptide can be directly recovered from the medium. If the polypeptide is not secreted into the medium, it can be recovered from cell lysates.
  • the polypeptide can be detected by methods known in the art that are specific to the polypeptide. These detection methods may include the use of specific antibodies, the formation of enzyme products, or the disappearance of enzyme substrates.
  • the produced polypeptide can be recovered by methods known in the art.
  • the cells can be harvested by centrifugation, broken up by physical or chemical methods, and the resulting crude extract is retained for further purification.
  • Any convenient method can be used to lyse the transformed host cells expressing the fusion protein of the present invention or its fragments, derivatives or analogs, including freeze-thaw cycles, ultrasound, mechanical disruption, or the use of cytolytic agents. These methods are well known to those skilled in the art.
  • the fusion protein of the present invention or its fragments, derivatives or analogues can be recovered and purified from the culture of transformed host cells.
  • the methods used include ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, and phosphocellulose. Chromatography, Hydrophobic Interaction Chromatography, Affinity Chromatography, Hydroxyapatite Chromatography, Phytohemagglutinin Chromatography, etc.
  • the nucleic acid encoding the fusion protein of the present invention can encode the amino acid sequence shown in SEQ ID NO: 9, and preferably has the nucleotide sequence shown in SEQ ID NO: 10.
  • the present invention also includes at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% with the preferred nucleic acid sequence of the present invention (SEQ ID NO: 10) , At least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence homology nucleic acid .
  • “Homology” or “identity” refers to the matching of sequences between two polypeptides or between two nucleic acids. When a certain position in the two sequences to be compared is occupied by the same base or amino acid monomer subunit (for example, a certain position in each of the two DNA molecules is occupied by adenine, or two A certain position in each of the polypeptides is occupied by lysine, so each molecule is the same at that position.
  • the "percent identity” between two sequences is a function of the number of positions compared by the number of matching positions shared by the two sequences x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity.
  • the nucleotide sequence in SEQ ID NO: 10 can be substituted, deleted or added one or more to generate a derivative sequence of SEQ ID NO: 10.
  • NO:10 has low homology, and can basically encode the amino acid sequence shown in SEQ ID NO:9.
  • “the nucleotide sequence in SEQ ID NO: 10 has been substituted, deleted, or added at least one nucleotide-derived sequence” means that it can be used under moderately stringent conditions, and more preferably under highly stringent conditions.
  • the nucleotide sequence to which the nucleotide sequence shown in SEQ ID NO: 10 hybridizes.
  • variant forms include (but are not limited to): deletion of several (usually 1-90, preferably 1-60, more preferably 1-20, and most preferably 1-10) nucleotides , Insertion and/or substitution, and adding several at the 5'and/or 3'end (usually within 60, preferably within 30, more preferably within 10, most preferably within 5 ) Nucleotide.
  • the polynucleotide or nucleic acid sequence of the present invention may be in the form of DNA or RNA.
  • the form of DNA includes: DNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be a coding strand or a non-coding strand.
  • polynucleotide encoding the fusion protein of the present invention may include a polynucleotide encoding the fusion protein, or a polynucleotide that also includes additional coding and/or non-coding sequences.
  • the present invention also relates to variants of the aforementioned polynucleotides, which encode fragments, analogs and derivatives of polyglycosides or polypeptides having the same amino acid sequence as the present invention.
  • the variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants and insertion variants.
  • allelic variant is an alternative form of a polynucleotide. It may be a substitution, deletion or insertion of one or more nucleotides, but it will not substantially change the function of the encoded polypeptide. .
  • the present invention also relates to polynucleotides that hybridize with the aforementioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences.
  • the present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions.
  • stringent conditions refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) adding during hybridization There are denaturants, such as 50% (v/v) methylphthalamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90% or more, It is more preferable that the hybridization occurs when more than 95%.
  • the full-length nucleic acid sequence of the present invention or its fragments can usually be obtained by PCR amplification method, recombination method or artificial synthesis method.
  • primers can be designed according to the relevant nucleotide sequence disclosed in the present invention, especially the open reading frame sequence, and a commercially available DNA library or a cDNA prepared by a conventional method known to those skilled in the art can be used.
  • the library is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.
  • the recombination method can be used to obtain the relevant sequence in large quantities. It is usually cloned into a vector, and then transferred into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.
  • artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is short. Usually, by first synthesizing multiple small fragments, and then ligating to obtain fragments with very long sequences.
  • the DNA sequence encoding the protein (or fragment or derivative thereof) of the present invention can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention through chemical synthesis.
  • the present invention provides a fusion protein and its coding sequence for highly efficient and site-specific removal of DNA methylation modification, which is of great significance for studying the function of DNA methylation.
  • the present invention provides for the first time the application of demethylated fusion protein in plants, and found that it has precise and efficient demethylation modification efficiency in plants, which is useful for studying epigenetics of plants and through demethylation regulation Plant traits have important scientific value.
  • sgRNA design five sgRNAs targeted to the MEMS region are designed.
  • the corresponding sgRNA sequences are shown in Table 1.
  • sgRNA also has a sticky end for ligation.
  • sgMEMS-1 and sgMEMS-4 are connected to U6 carrier; sgMEMS-2 and sgMEMS-5 are connected to U3b carrier; sgMEMS-3 is connected to 7SL carrier. Sequencing verified that the sgRNA was successfully ligated into the corresponding vector.
  • the three mixed sgRNA fragments obtained above were ligated to the p1300-UBQ-dCas9-TET1cd and p1300-UBQ-dCas9-ROS1cd vectors respectively using T4 ligase.
  • the p1300-sgMEMS1_2_3-UBQ-dCas9-TET1cd and p1300-sgMEMS1_2_3-UBQ-dCas9-ROS1cd vector can be obtained after the ligation reaction at 16°C for 2h. Sequencing verifies that the fragments are correctly connected to the vector.
  • p1300-sgMEMS1_2_3-UBQ-dCas9-TET1cd and p1300-sgMEMS1_2_3-UBQ-dCas9-ROS1cd vectors were digested with Kpn I+EcoR I, purified and recovered by ethanol; the mixed sgRNA fragments were connected into p1300-sgMEMS1_2_3 with T4 ligase -UBQ-dCas9-TET1cd and p1300-sgMEMS1_2_3-UBQ-dCas9-ROS1cd carrier, the final carrier p1300-sgMEMS1_2_3-UBQ-dCas9-TET1cd-sgMEMS4_5 and p1300-sgMEMS1_2_3-UBQ-dCas9-ROS1cd-sg-sg4 carrier.
  • DNA extraction uses QIAGEN's plant DNA extraction kit.
  • Control DNA methylation/positive seedling methylation>1.5 is a positive seedling that is successfully edited.
  • One plant is selected from the plant with vector and one plant without the vector, and the leaves are taken again, and DNA is extracted with QIAGEN's plant DNA extraction kit.
  • dCas9-ROS1cd and dCas9-TET1cd reduce the methylation level of MEMS in the target region in transgenic T1 plants
  • transgenic plants No. 13 and No. 14 of dCas9-ROS1cd, No. 5 and No. 14 transgenic plants of dCas9-TETcd1 were significantly demethylated compared to the wild-type and dCas9 positive control plants. Retouch.
  • the transgenic line with dCas9-TET1cd maintained the original low methylation level at the MEMS site, and the dCas9-TET1cd T2 individuals without the transgene showed methylation reversal.
  • Both dCas9-ROS1cd and dCas9-TET1cd can mediate the demethylation of MEMS sites in the ROS1 promoter region in plants, and the demethylation editing efficiency of dCas9-ROS1cd is higher than that of dCas9-TET1cd.
  • Demethylation of MEMS sites can effectively reduce the expression of ROS1 gene. It shows that DNA methylation and demethylation can effectively regulate gene expression.
  • sgRNAs 1, 2, and 3 are connected to the upstream of the fusion protein, while sgRNAs 4, 5, and 6 are connected to the downstream of the fusion protein.
  • the sequence of sgRNA is shown in Table 3. sgRNA is consistent.
  • T4 ligase Use T4 ligase to ligate the recovered fragments into Takara's p20T vector.
  • a, b, and c respectively correspond to the methylation editing results of 3 sites; the bottom of each figure represents the position of the editing region on the chromosome, the red line represents the position of the CG site on the genome, and the blue The line represents the position of the CHG site on the genome, the black arrow represents the position of the primer used to analyze DNA methylation, and the position of the sgRNA corresponding to the genome is also marked in the figure; the top of each figure represents the level of DNA methylation , The solid represents DNA methylation at the corresponding site, the open represents no DNA methylation, red represents CG methylation, blue represents CHG methylation, and green represents CHH methylation.
  • the T2 individuals with and without the transgene maintained their hypomethylated state.
  • Both dCas9-ROS1cd and dCas9-TET1cd can mediate the demethylation of DNA at Chr4.8670151-8671193, and the demethylation editing efficiency of dCas9-TET1cd is higher than that of dCas9-ROS1cd.
  • the Chr5.9872445-9873033 (solo-LTR site) site
  • only dCas9-ROS1cd successfully demethylated it.
  • Chr3:2849440-2849791 only dCas9-TET1cd successfully demethylated it.
  • dCas9-ROS1cd and dCas9-TET1cd show different efficiencies, and they can complement each other when applied.

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Abstract

L'invention concerne une protéine de fusion et son utilisation. Plus particulièrement, l'invention concerne une protéine de fusion, qui comprend un composant choisi parmi les composants suivants, ou est composé des composants suivants : (1) Un élément fonctionnel de positionnement D1, qui a la fonction de cibler et de lier l'ADN ; et (2) un élément fonctionnel déméthylé D2, qui a pour fonction de convertir un nucléotide méthylé en un nucléotide non méthylé.
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