WO2021169925A1

WO2021169925A1 - Fusion protein and application thereof

Info

Publication number: WO2021169925A1
Application number: PCT/CN2021/077328
Authority: WO
Inventors: 牛小牧; 李彦莎; 梁亚峰
Original assignee: 山东舜丰生物科技有限公司
Priority date: 2020-02-26
Filing date: 2021-02-23
Publication date: 2021-09-02
Also published as: CN113307878A

Abstract

Provided are a fusion protein and an application thereof. Specifically, provided is a fusion protein, which comprises a component selected from among the following, or is composed of the following components: (1) a positioning functional element D1, which has the function of targeting and binding DNA; and (2) a demethylated functional element D2, which has the function of converting a methylated nucleotide into an unmethylated nucleotide.

Description

A fusion protein and its application

Technical field

The invention belongs to the field of biotechnology, and specifically relates to a fusion protein and its application.

Background technique

DNA methylation (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.

There are many ways to modify the methylation. 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. There are two mechanisms for DNA demethylation: passive demethylation and active demethylation. 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.

Studies have shown that DNA demethylation plays an important role in the reactivation of silent genes. In addition, DNA methylation can lead to changes in DNA conformation in certain regions, thereby affecting the interaction between protein and DNA, leading to gene silencing. In plants, DNA methylation controls a variety of biological processes, including flower morphology, sex determination, plant structure, flowering time, biomass, and leaf senescence. By controlling DNA methylation, 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.

Therefore, there is an urgent need in this field to develop a method that can efficiently and site-specifically methylate or demethylate DNA.

Summary of the invention

The present invention provides a protein capable of demethylating and modifying nucleic acid efficiently and site-specifically and its application.

In the first aspect of the present invention, a fusion protein is provided, comprising components selected from the following:

(1) The positioning function element D1, which has the function of targeting and binding DNA; and

(2) Demethylation functional element D2, which has the function of converting methylated nucleotides into non-methylated nucleotides.

In another preferred embodiment, 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.

In another preferred embodiment, the D1 element is selected from the following group: dCas9, dCpf1, dCas12, dCas13, dCms1, dMAD7, or functional domains thereof, or combinations thereof.

In another preferred embodiment, the D1 element is dCas9.

In another preferred example, the D1 element comprises a sequence selected from the following, or consists of a sequence selected from the following:

(1) The amino acid sequence shown in SEQ ID NO: 1;

(2) Compared with the sequence shown in SEQ ID NO: 1, there are one or more amino acid substitutions, deletions or additions (e.g. 1, 2, 3, 4, 5, 6, 7, etc.) 8, 9, or 10 amino acid substitutions, deletions or additions) sequence; or

(3) The 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.

In another preferred embodiment, the D2 element has the function of converting methylated cytosine into unmethylated cytosine.

In another preferred example, 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.

In another preferred embodiment, the D2 element is ROS1 or its functional domain.

In another preferred example, the D2 element comprises a sequence selected from the following, or consists of a sequence selected from the following:

(1) The amino acid sequence shown in SEQ ID NO: 3;

(2) The sequence shown in 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

(3) The 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.

In another preferred example, the D1 element is located at the N end or C end of the D2 element.

In another preferred example, 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.

In another preferred example, the nuclear localization signal comprises a sequence selected from the following, or consists of a sequence selected from the following:

(1) The amino acid sequence shown in SEQ ID NO: 5 or SEQ ID NO: 7;

(2) Compared with the sequence shown in SEQ ID NO: 5 or SEQ ID NO: 7, there are one or more amino acid substitutions, deletions or additions (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions) sequence; or

(3) The 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.

In another preferred example, 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.

In another preferred example, the fusion protein comprises a sequence selected from the following, or consists of a sequence selected from the following:

(1) The amino acid sequence shown in SEQ ID NO: 9;

(2) Compared with the sequence shown in SEQ ID NO: 9, there are one or more amino acid substitutions, deletions or additions (e.g. 1, 2, 3, 4, 5, 6, 7, etc.) 8, 9, or 10 amino acid substitutions, deletions or additions) sequence; or

(3) The 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.

In another preferred embodiment, 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.

In the second aspect of the present invention, a fusion protein combination is provided, the fusion protein combination 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;

In another preferred embodiment, 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-(X)n-D2 (Formula I)

Where

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 represents an integer of 0-6;

"-" indicates a peptide bond connecting the above-mentioned elements;

Among them, the position order of D1 and D2 can be interchanged;

In addition, D2 in the respective structures of the first fusion protein and the second fusion protein are different.

When n is 0, D1 and D2 are directly connected by peptide bonds.

In another preferred example, n is 1.

In another preferred example, X is a nuclear localization signal.

In another preferred example, D2 of the first fusion protein is ROS1 or its functional domain; D2 of the second fusion protein is TET1 or its functional domain.

In the third aspect of the present invention, there is provided a nucleic acid encoding the fusion protein according to the first aspect of the present invention.

In another preferred example, the sequence of the nucleic acid includes the following elements:

(1) Z1 is the nucleotide sequence encoding the positioning function element D1 in the fusion protein; and

(2) Z2 is the nucleotide sequence encoding the functional demethylation element D2 in the fusion protein.

In another preferred example, the Z1 element comprises a sequence selected from the following, or consists of a sequence selected from the following:

(i) SEQ ID NO: the sequence shown in 2;

(ii) Compared with the sequence shown in SEQ ID NO: 2, there are substitutions, deletions or additions of one or more bases (e.g. 1, 2, 3, 4, 5, 6, 7 , 8, 9, or 10 base substitution, deletion or addition) sequence;

(iii) The 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;

(iv) A sequence that hybridizes to the sequence described in any one of (i) to (iii) under stringent conditions; or

(v) The reverse complement of the sequence described in any one of (i) to (iii).

In another preferred example, the Z2 element comprises a sequence selected from the following, or consists of a sequence selected from the following:

(i) SEQ ID NO: the sequence shown in 4;

(ii) Compared with the sequence shown in SEQ ID NO: 4, there are substitutions, deletions or additions of one or more bases (e.g. 1, 2, 3, 4, 5, 6, 7 , 8, 9, or 10 base substitution, deletion or addition) sequence;

(iii) The 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;

In another preferred example, the nucleic acid comprises a sequence encoding a nuclear localization signal, and the sequence encoding the nuclear localization signal has a sequence selected from the following, or consists of a sequence selected from the following:

(i) The sequence shown in any one of SEQ ID NO: 6 or SEQ ID NO: 8;

(ii) Compared with the sequence shown in any one of SEQ ID NO: 6 or SEQ ID NO: 8 with one or more base substitutions, deletions or additions (for example, 1, 2, 3, 4 , 5, 6, 7, 8, 9, or 10 base substitutions, deletions or additions);

(iii) The 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;

In another preferred example, the nucleic acid has a sequence selected from the following, or consists of a sequence selected from the following:

(i) SEQ ID NO: the sequence shown in 10;

(ii) Compared with the sequence shown in SEQ ID NO: 10, there are substitutions, deletions or additions of one or more bases (e.g. 1, 2, 3, 4, 5, 6, 7 , 8, 9, or 10 base substitution, deletion or addition) sequence;

(iii) The 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;

In the fourth aspect of the present invention, there is provided a 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.

In another preferred embodiment, the 5 ^'and / or ^3' of the end of the first nucleic acid sequence comprises one or more nuclear localization signal.

In another preferred embodiment, 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.

In another preferred embodiment, the nucleic acid construct contains 1-6 gRNA coding sequences.

In another preferred embodiment, the coding sequence of the gRNA series distribution at the 5 ^'end or ^3' end of the first nucleic acid sequence.

In another preferred example, when two or more second nucleic acid sequences are contained, the second nucleic acid sequences are distributed at both ends of the first nucleic acid structure sequence.

In another preferred example, the 5'end of 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.

In the fifth aspect of the present invention, a vector is provided, 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.

In the sixth aspect of the present invention, there is provided a composite comprising:

(1) 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.

(2) The nucleic acid component is one or more gRNA sequences;

Wherein, the protein component and the nucleic acid component combine with each other to form the complex.

In the seventh aspect of the present invention, a polynucleotide combination is provided, which encodes the fusion protein combination according to the second aspect of the present invention.

In another preferred embodiment, 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.

In another preferred embodiment, the first nucleotide and the second nucleotide respectively further include one or more gRNA coding sequences.

In another preferred embodiment, the first polynucleotide and the second nucleotide are located in the same vector or different vectors.

In another preferred embodiment, the first polynucleotide and the second nucleotide are located in different vectors.

In another preferred embodiment, the vector containing the first nucleic acid and the vector containing the second nucleic acid transform cells simultaneously or sequentially.

In the eighth aspect of the present invention, there is provided 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.

In another preferred embodiment, the host cell is a eukaryotic cell or a prokaryotic cell.

In another preferred embodiment, the host cell is a plant cell.

In another preferred example, 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:

(1) expressing the host cell according to the eighth aspect of the present invention under suitable conditions,

(2) Separate and extract the fusion protein.

In another preferred embodiment, 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.

In the tenth aspect of the present invention, 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.

In another preferred embodiment, the demethylation is the conversion of methylated cytosine to unmethylated cytosine.

In another preferred example, the target nucleic acid is derived from a eukaryote or a prokaryote.

In another preferred embodiment, the target nucleic acid is derived from plant cells or animal cells.

In another preferred embodiment, the target nucleic acid is derived from the nucleus, cytoplasm, chloroplast or mitochondria.

In another preferred embodiment, the target nucleic acid is DNA, RNA or a combination thereof.

In the eleventh aspect of the present invention, 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.

In the twelfth aspect of the present invention, a kit is provided, 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 The polynucleotide combination described in this aspect and the host cell described in the eighth aspect of the present invention.

In the thirteenth aspect of the present invention, there is provided a method for reducing DNA methylation of a target gene or its promoter or its enhancer in a cell. The method 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.

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.

In another preferred embodiment, the regulation includes: activation, enhancement, inhibition, reduction or inactivation.

In another preferred embodiment, 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.

In another preferred example, the expression control elements include: promoter, enhancer, terminator, transposon, and silencer.

In the fifteenth aspect of the present invention, a method for regulating plant traits is provided, which is characterized by comprising the following steps:

(i) Provide a plant cell;

(ii) The 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;

(iii) culturing the cells into seedlings;

(iv) Screening plants with target traits.

In another preferred embodiment, the method for introducing cells includes Agrobacterium infection, gene gun transformation, microinjection, electric shock, ultrasound, and polyethylene glycol (PEG)-mediated method.

In another preferred embodiment, the traits are epigenetic traits of plants.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as the embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, I will not repeat them one by one here.

Description of the drawings

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.

Detailed ways

After extensive and in-depth research and a large number of screenings, 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. Experiments show that 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.

the term

As used herein, the term "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.

As used herein, the term "fusion protein combination" refers to a combination of multiple fusion proteins in the present invention. In the fusion protein combination of the present invention, each fusion protein has a different demethylase catalytic domain. Preferably, the different demethylase catalytic domains have different demethylation effects on different target nucleic acid sites, so that they complement each other.

As used herein, the term "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. In the present invention, 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.

As used herein, the term "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. .

As used herein, 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. For example, the commonly used connecting peptide can be XTEN.

As used herein, the "gRNA" is also called guide RNA or guide RNA, and has the meaning commonly understood by those skilled in the art. Generally speaking, 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. In different CRISPR systems, 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. In some cases, 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. In certain embodiments, when optimally aligned, 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.

As used herein, 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.

As used herein, the term "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. Currently discovered 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.

Demethylase

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. Studies have shown that demethylation modification has an important correlation with processes such as imprinted gene expression, fruit development, biotic and abiotic stress, nodule development, and nodule nitrogen fixation in plant growth and development. 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.

Fusion protein of the present invention and its coding sequence

The present invention provides a fusion protein, which has the function of targeted binding to DNA and converting target methylated nucleotides into unmethylated nucleotides.

Wherein, 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. For example, the D1 element is selected from the following group: dCas9, dCpf1, dCas12, dCas13, dCms1, dMAD7, or a combination thereof. Preferably, the D1 element is dCas9.

In a preferred embodiment, 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.

Preferably, the D2 element has the function of converting methylated cytosine into unmethylated cytosine. For example, 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.

In a preferred embodiment, 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.

In another preferred example, 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. Preferably, 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.

In a particularly preferred embodiment, the fusion protein comprises a sequence selected from the following, or consists of a sequence selected from the following:

(1) The amino acid sequence shown in SEQ ID NO: 9;

In another preferred embodiment, 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.

The present invention also includes fragments and analogs having the functions of the fusion protein of the present invention. As used herein, the terms "fragment" and "analog" 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). According to the definition herein, these fragments, derivatives and analogs belong to the scope well known to those skilled in the art.

In the present invention, 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. For example, in the protein, when 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. These conservative variants are best generated according to Table A by substitution.

Table A

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 β, γ-amino acids). It should be understood that the protein of the present invention is not limited to the representative proteins exemplified above.

Modifications (usually without changing the primary structure) 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.

In the production method of the present invention, 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. For example, 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.

In a particularly preferred embodiment, 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. Usually, when it is difficult to compare two sequences to produce the maximum identity, such an alignment can be performed by using, for example, Needleman, etc., which is conveniently performed by a computer program such as the Align program (DNAstar, Inc.) (DNAstar, Inc.) Human (1970) J. Mol. Biol. J. Mol. Biol. J. Mol. Biol. J. Mol. Biol. 48: 443-453. You can also use the algorithms of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) that have been integrated into the ALIGN program (version 2.0), and use the PAM120 weight residue table (weight residue table) A gap length penalty of 12 and a gap penalty of 4 are used to determine the percent identity between two amino acid sequences. This method can be achieved. In addition, you can use the Needleman and Wunsch (J MoI Biol.48:444-453(1970)) algorithms in the GAP program integrated into the GCG software package (available on www.gcg.com), use the Blossum 62 matrix or PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two amino acid sequences . Herein, variants of the gene can be obtained by inserting or deleting regulatory regions, performing random or site-directed mutations, and the like.

In the present invention, 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. In addition, "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. These 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.

The term "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. As known in the art, an 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. In the present invention, "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. For the PCR amplification 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. Once the relevant sequence is obtained, 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.

In addition, 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. At present, 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 main advantages of the present invention include:

1) 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.

2) 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.

The present invention will be further explained below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following examples usually follow conventional conditions, such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions described in the manufacturing The conditions suggested by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and parts by weight.

Example 1: Construction of demethylated gene editing tool vector

1.1 dCas9-TET1cd demethylation tool

(1) Using the existing dCas9 and TET1 sequences in the laboratory, the high-fidelity enzyme Q5 was used to amplify the sequence fragments of dCas9 and TET1cd. Glue recovery fragments for future use.

(2) The p1300-UBQ-CAS9 vector cut with Nco I and BamH I, and the p1300-UBQ fragment is recovered by cutting the gel, for use.

(3) Use recombinase to recombine the dCas9 fragment into the p1300-UBQ fragment to obtain p1300-UBQ-dCas9 for use. Sanger sequencing was used to prove that the fragments were successfully recombined.

(4) Next, the p1300-UBQ-dCas9 vector obtained above was cut with BamHI.

(5) Using recombinase to recombine the TET1cd fragment into the p1300-UBQ-dCas9 fragment to obtain the p1300-UBQ-dCas9-TET1cd vector, which is the final vector for targeted editing of DNA methylation. Sanger sequencing was used to prove that the fragments were successfully recombined.

1.2 dCas9-ROS1cd demethylation tool

(1) Amplify the ROS1cd sequence using Arabidopsis cDNA.

(2) Use recombinase to recombine the ROS1cd fragment into the p1300-UBQ-dCas9 fragment to obtain the p1300-UBQ-dCas9-ROS1cd vector, which is the final vector for targeted editing of DNA methylation. Sanger sequencing was used to prove that the fragments were successfully recombined.

Example 2: Regulation of ROS1 expression by MEMS demethylation in the ROS1 promoter region

2.1 Target design and construction

(1) According to the rules of sgRNA design, five sgRNAs targeted to the MEMS region are designed. The corresponding sgRNA sequences are shown in Table 1. In addition to the 20bp targeted to the MEMS region, sgRNA also has a sticky end for ligation.

Table 1 sgRNA sequence targeting MEMS region

(2) Turn the F and R sequences of sgRNA into double-stranded DNA fragments with sticky ends through annealing program. The process is: dilute the positive and negative primers to 100μM, take 1μL each with 1μL T4 DNA ligase buffer, 0.5μL T4 polynucleotide kinase, 6.5μL ddH2O, mix the mixture at 37°C for 30min, 95°C for 5min, and then The rate of 0.2°C/s was reduced to 25°C, and finally it was diluted 250 times with water.

(3) U6, U3b, and 7SL vectors are digested with Bbs I, and the vector fragments are recovered for use.

(4) Take 1 μL of double-stranded sgRNA fragment and 1 μL of digested vector, and use T4 ligase to connect sgRNA into U6, U3b, and 7SL vectors. 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.

(5) Amplify the U6-sgMEMS-1, U3b-sgMEMS-2, and 7SL-sgMEMS-3 carriers obtained above with the corresponding primers, and recover the fragments with the promoter and sgRNA by gel recovery, and use them for later use.

(6) Use Sbf I+Xho I, Xho I+Xba I, Xba I+Xma I to digest the U6-sgMEMS-1, U3b-sgMEMS-2, and 7SL-sgMEMS-3 fragments obtained above, and mix and purify them. Recycle, spare.

(7) Use Sbf I+Xma I to digest the p1300-UBQ-dCas9-TET1cd and p1300-UBQ-dCas9-ROS1cd vectors, and recover by ethanol precipitation for use.

(8) 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.

(9) Next, connect sgMEMS4 and sgMEMS5 to p1300-sgMEMS1_2_3-UBQ-dCas9-TET1cd and p1300-sgMEMS1_2_3-UBQ-dCas9-ROS1cd carrier, the method is similar to the above. First, the U6-sgMEMS4 and U3b-sgMEMS5 fragments were obtained by PCR amplification with corresponding primers; then, the U6-sgMEMS4 and U3b-sgMEMS5 fragments obtained by digestion with Kpn I+Xho I and Xho I+EcoR I, respectively, were mixed and purified by column purification. ; Then, 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.

2.2 Genetic transformation

(1) The p1300-sgMEMS1_2_3-UBQ-dCas9-TET1cd-sgMEMS4_5 and p1300-sgMEMS1_2_3-UBQ-dCas9-ROS1cd-sgMEMS4_5 vectors were directly transformed into Agrobacterium GV3101.

a. Add the plasmid to Agrobacterium competent cells, then ice bath for 5 min, then put it in liquid nitrogen for 5 min, and then 37°C water bath for 5 min.

b. Take out the centrifuge tube, add an appropriate amount of antibiotic-free LB broth (500μL), shake culture at 28°C for 2h.

c. Take a small amount of bacterial liquid (50μL) and smear it on the solid LB medium resistant to kanaampicin and rifampicin, and incubate it in an incubator at 28°C for 2 days, and the colonies can be seen to grow.

(2) Transform the Agrobacterium carrying the carrier into Arabidopsis thaliana.

a. Pick 3 monoclonal colonies obtained above and place them in LB culture medium containing 3 mL of the corresponding antibiotics, and culture them with shaking at 28°C for 16 hours.

b. Remove 1 mL of the above-mentioned bacterial liquid and incubate overnight in LB culture medium containing 100 mL of corresponding antibiotics, and determine the OD value of 1.5-2.0.

c. Centrifuge at 4000g for 10min at room temperature to collect Agrobacterium cells, and resuspend the Agrobacterium in a newly prepared 100mL 5% sucrose solution.

d. Add 20μL Silwet L-77 to the above sucrose suspension bacteria liquid.

e. The above-ground part of flowering Arabidopsis is immersed in the above solution for about 15 seconds. Wrap it with plastic wrap and place it in a black tray, protect it from light, and place it in a greenhouse. After 16-24 hours, take it out for normal culture.

f. When the fruit pods turn yellow, collect the seeds as T1 seeds, and set aside.

2.3 Screening of transgenic positive vaccines

(1) Disinfect and sterilize the T1 seeds with 5% sodium hypochlorite solution, and wash them with sterile water 5 times before use.

(2) Resuspend the seeds in an appropriate amount of sterile water, and then pour them on 1/2MS medium containing hygromycin, so that the seeds are evenly distributed on the medium, and after drying, wrap the board with tin foil , Placed in a refrigerator at 4°C for 7 days.

(3) Put the board in a constant temperature incubator for 10-14 days. Transplant the positive seedlings into the soil and place them in the greenhouse for cultivation.

2.4 Detection of DNA methylation level and editing efficiency

(1) When the positive seedlings grow to a suitable size, take the leaves of the positive seedlings to extract DNA. DNA extraction uses QIAGEN's plant DNA extraction kit.

(2) Determine the methylation level of positive seedlings

a. Treat the DNA of the positive seedlings with bisulfite. This step is completed with the kit named BisulFlash DNA Modification.

b. Amplify the above-mentioned processed DNA with specially designed primers specially used for methylation sequencing, and gel to recover the DNA for use.

c. Mix the gel recovery products corresponding to each positive seedling, and then send them to the sequencing platform for sequencing.

d. Analyze methylation data and count the efficiency of methylation editing. We define: Control DNA methylation/positive seedling methylation>1.5 is a positive seedling that is successfully edited.

2.5 Genetic stability of DNA demethylation

(1) Select a successfully edited positive seedling, and harvest the seed as T2.

(2) Disinfect and sterilize the harvested T2 seeds, plant them on a normal 1/2 MS medium, place them at 4°C for 7 days, place them in a constant temperature incubator for 14 days, and transplant them into the soil for cultivation in a greenhouse.

(3) Select several plants, take their leaves, extract their DNA with CTAB method, and use M13F and sgRNA analysis to identify plants with and without vectors.

(4) 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.

(5) Analyze the level of DNA methylation.

2.6 Gene expression analysis

(1) For selected plants, take their leaves and extract RNA with QIAGEN's plant RNA extraction kit.

(2) Reverse RNA into cDNA with a full gold reverse transcription kit.

(3) Analyze the expression level of genes with Takara's SYBR.

2.7 Experimental results

(1) dCas9-ROS1cd and dCas9-TET1cd reduce the methylation level of MEMS in the target region in transgenic T1 plants

As shown in Figure 1, 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.

(2) Expression level of ROS1

As shown in Figure 2, the expression levels of ROS1 in the transgenic plants No. 13 and 14 of dCas9-ROScd1, and the transgenic plants No. 5 and 14 of dCas9-TET1cd were lower than those of the wild type and the control group.

(3) Editing efficiency of dCas9-ROS1cd and dCas9-TET1cd at MEMS sites

Table 2

(4) Genetic stability of MEMS site demethylation

As shown in Figure 3, among the T2 generation plants, 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.

2.8 Experimental conclusion

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.

Example 3: Demethylation experiment in RdDM mutant (nrpd1)

3.1 Target design

The target design is consistent with the previous one. 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.

Table 3 sgRNA sequences targeting multiple regions

3.2 Genetic transformation

See step 2 genetic transformation process in the experimental example.

3.3 Screening of positive vaccines

See step 3 in the experimental example for positive vaccine screening.

3.4 Detection of DNA methylation level and editing efficiency

(1) When the positive seedlings grow to a suitable size, take the leaves of the positive seedlings and extract DNA with the CTAB method.

(2) Use Chop-PCR to analyze the methylation level of positive seedlings.

a. Treat 1ug of DNA with appropriate methylation-sensitive restriction enzymes for 12h-16h.

b. Use the corresponding primers to amplify the digested DNA, electrophoresis, and judge the level of methylation based on the light and dark bands.

(3) Mark the positive seedlings with reduced methylation judged by Chop-PCR, take the leaves again, and extract DNA with GIAGEN kit.

(4) Methylation sequencing to analyze DNA methylation level

a. Treat the DNA extracted from the kit with bisulfite.

b. Use the designed primers to expand the processed DNA, electrophoresis, gel recovery, and use it for later use.

c. Use T4 ligase to ligate the recovered fragments into Takara's p20T vector.

d. Pick positive clones, colony PCR, and sequencing.

e. Analyze the methylation level with KISMETH.

f. Use Chop-PCR to count the methylation editing efficiency.

3.5 Genetic stability of DNA demethylation

Consistent with the previous, only the enzyme-linked sequencing analysis was used to determine the methylation.

3.6 Experimental results

(1) Results of demethylation of different regions by dCas9-ROS1cd and dCas9-TET1cd

Legend: 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.

As shown in Figure 4, at Chr4.8670151-8671193, the L44 transgenic plants of dCas9-ROS1cd and the L4 transgenic plants of dCas9-TETcd1 have undergone significant demethylation modification, and almost all of their DNA methylation has been targeted. To remove. At Chr5.9872445-9873033 (solo-LTR site) site, only dCas9-ROS1cd J41 transgenic plants had significant demethylation modification. In contrast, at Chr3:2849440-2849791, only E6 of dCas9-TET1cd was significantly demethylated.

(2) Editing efficiency of dCas9-ROS1cd and dCas9-TET1cd in different regions

Table 4

(3) Genetic stability

As shown in Figure 5, in the T2 generation plants, the T2 individuals with and without the transgene maintained their hypomethylated state.

3.7 Experimental conclusion

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. However, for the Chr5.9872445-9873033 (solo-LTR site) site, only dCas9-ROS1cd successfully demethylated it. In contrast, for Chr3:2849440-2849791, only dCas9-TET1cd successfully demethylated it. For different sites, dCas9-ROS1cd and dCas9-TET1cd show different efficiencies, and they can complement each other when applied.

All documents mentioned in the present invention are cited as references in this application, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

A fusion protein characterized by comprising components selected from the following:

(1) The positioning function element D1, which has the function of targeting and binding DNA; and

(2) Demethylation functional element D2, which has the function of converting methylated nucleotides into non-methylated nucleotides.
The fusion protein of claim 1, wherein the D1 has no catalytic activity and is selected from the group consisting of Cas protein, zinc finger protein or TALENs protein, or functional domains thereof, or a combination thereof;

Preferably, the D1 is selected from the following group: dCas9, dCpf1, dCas12, dCas13, dCms1, dMAD7, or functional domains or combinations thereof.
The fusion protein of claim 1 or 2, wherein the D2 has the function of converting methylated cytosine to unmethylated cytosine;

Preferably, the D2 is a demethylase or its demethylation functional domain selected from the following group: ROS1, TET, DME, DML, or a combination thereof.
The fusion protein of claim 1, wherein the D1 and D2 are connected by one or more of the following components: peptide bond, connecting peptide, nuclear localization signal, epitope tag, or a combination thereof.
A fusion protein combination, characterized in that the fusion protein combination includes a first fusion protein and a second fusion protein;

The structures of the first fusion protein and the second fusion protein are each independently as shown in the fusion protein of any one of claims 1 to 4;

And D2 in the first fusion protein and the second fusion protein are different;

Preferably, D2 of the first fusion protein is selected from ROS1 or its functional domain, and D2 of the second fusion protein is selected from TET or its functional domain.
A nucleic acid characterized in that it encodes the fusion protein according to any one of claims 1 to 4 or the fusion protein combination according to claim 5.
A nucleic acid construct, which is characterized by comprising a first nucleic acid sequence and one or more second nucleic acid sequences, wherein the first nucleic acid sequence encodes the fusion protein or the right according to any one of claims 1 to 4 The fusion protein combination of claim 5, wherein the second nucleic acid sequence is a gRNA coding sequence.
A vector, characterized in that it contains the nucleic acid of claim 6 or the nucleic acid construct of claim 7.
A compound characterized in that it contains:

(1) A protein component, comprising the fusion protein according to any one of claims 1 to 4 or the fusion protein combination according to claim 5.

(2) The nucleic acid component is one or more gRNA sequences;

Wherein, the protein component and the nucleic acid component combine with each other to form the complex.
A host cell, characterized in that the host cell contains the fusion protein according to any one of claims 1 to 4, or the fusion protein combination according to claim 5, or the vector according to claim 8, Or the complex of claim 9, or the nucleic acid of claim 6 or the nucleic acid construct of claim 7 integrated into the genome of the host cell.
The fusion protein of any one of claims 1-4, or the fusion protein combination of claim 5, or the nucleic acid of claim 6, or the nucleic acid construct of claim 7, or the nucleic acid construct of claim 8 Use of the vector or the complex of claim 9 in the demethylation modification of target nucleic acid.
The fusion protein of any one of claims 1-4, or the fusion protein combination of claim 5, or the nucleic acid of claim 6, or the nucleic acid construct of claim 7, or the nucleic acid construct of claim 8 Use of the vector or the complex of claim 9 in the preparation of a kit for demethylation modification of a target nucleic acid.