WO2023286891A1 - A method for tracking transposition of genetic material - Google Patents

A method for tracking transposition of genetic material Download PDF

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WO2023286891A1
WO2023286891A1 PCT/KR2021/009179 KR2021009179W WO2023286891A1 WO 2023286891 A1 WO2023286891 A1 WO 2023286891A1 KR 2021009179 W KR2021009179 W KR 2021009179W WO 2023286891 A1 WO2023286891 A1 WO 2023286891A1
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fluorescent protein
nucleic acid
acid molecule
truncated
seq
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French (fr)
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Jungnam CHO
Eun Yu Kim
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Cas Center For Excellence In Molecular Plant Sciences
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1082—Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof

Definitions

  • the present invention relates to a recombinant transposable element and a method for detecting and tracking a transpositional issue in an organism using the same.
  • Transposable element is a stretch of DNA that can move from one position to another in the genome. Evolutionarily it has served as a major source of genetic variability, which is critical for the diversity and evolution of a species. On the other hand, transposons are also potentially dangerous because they can cause fatal mutations as they insert into genes. The importance of transposons in plant science has been growing recently as gene editing technologies are becoming more available in crop plants. The current gene editing methods exclusively require tissue culture techniques by which massive transposons can be activated, and therefore crop genome integrity can be damaged. Crop genomes are, in fact, even more vulnerable to these natural mutagens because they usually contain myriads of transposons by up to around 90%.
  • Transposon-imposed genomic instability is a considerable bottleneck to modern crop breeding, and therefore understanding their mobility is very important.
  • transposons the discovery of which dates back to the early 1940s, and its ever-growing importance in crop breeding, we are still far from understanding how they proliferate in the genome.
  • the present inventors have developed a ground-breaking technology to track transposon mobility in real-time and single-nuclei resolution.
  • the host regulators of transposon jumping are expected to be identified by coupling with the genome-wide genetic screening.
  • Genomic instability frequently happens during tissue culture, which is a widely-used agricultural practice to propagate crop plants and modify genetic information. Such unwanted and unpredicted genomic alteration hampers eventually food safety which is an immediate threat to people's health. For this reason, the present inventors aim to understand the mechanisms of transposons, the primary agents of genomic instability, for the purpose of making good control of them. To tackle this problem, the present inventors have been working to establish a ground-breaking method to observe their action accurately and to dissect the detailed process of their mobility.
  • nucleic acid molecule comprising:
  • the present inventors have made intensive studies to develop a real-time method to trace a transposition of genetic material in a biological sample or an organism. As results, the present inventors have discovered that a recombinant transposable element in which LTR regions have been replaced by truncated fluorescent protein gene may produce functional fluorescent signal only in case sequential reverse transcription reaction reconstitutes the incomplete fluorescent protein genes into the intact one upon the transcriptional activation by chemical-inducible promoter.
  • the novel recombinant transposable element suggested by the present inventors and tracing methods using the same efficiently overcome the limitations of the conventional method where transposon may be displayed in the following generations after the activation of transposable element mobility.
  • nucleic acid molecule as used herein has comprehensive meaning including DNA (gDNA and cDNA) and RNA molecule.
  • a nucleotide which is a basic construct unit of nucleic acid molecule, includes nucleotide analogues with modified sugar or base, as well as natural-occurring nucleotides (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584(1990)).
  • genetic material refers to any material storing genetic information in the nuclei or an organism's cells.
  • the genetic material includes DNA or RNA that is passed along from one generation to the next.
  • biological sample refers to not only samples that are obtainable from an organism, but also any samples that may contain genetic material.
  • examples of the biological sample include human and animal blood, plant body fluids, human and animal waste, microbial culture liquid, cell culture liquid, virus culture liquid, biopsy culture liquid, soil and air, but are not limited thereto.
  • regulatory sequence refers to a DNA sequences which are necessary to control the expression of sequences to which they are ligated.
  • the regulatory sequences may differ depending upon the intended host organism and upon the nature of the sequence to be expressed.
  • chemical-inducible regulatory sequence refers to a regulatory sequence, e.g., a promoter, that are activated by exposure to a specific chemical compound.
  • the chemical-inducible regulatory sequence is estradiol-inducible promoter sequence.
  • promoter refers to a region of DNA upstream from the transcription start and which is involved in binding RNA polymerase and other proteins to start transcription.
  • An Estradiol-inducible promoter enables selective initiation of transcription based on the existence of beta-estradiol in biological sample.
  • the 3'-truncated fluorescent protein and the 5'-truncated fluorescent protein are each independently selected from the group consisting of green fluorescent protein (GFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), blue fluorescent protein (BFP) and yellow fluorescent protein (YFP). More concretely, the 3'-truncated fluorescent protein and the 5'-truncated fluorescent protein are GFP.
  • truncated refers to a deletion of nucleotide or amino acid sequence to an extent where the biological activity of the full-length nucleotide or peptide may not be maintained. Therefore, any biologically equivalent variations are excluded from the “truncated protein" of the present invention.
  • the 3'-truncated fluorescent protein linked to the 3' end of the transposable element and the 5'-truncated fluorescent protein linked to the 5' end of the transposable element share at least 30 residues of the common amino acids, and more concretely, at least 40 residues of the common amino acids.
  • the 5'-truncated fluorescent protein comprises an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 90% homology, more concretely at least 95% homology, with SEQ ID NO: 1.
  • the nucleotide encoding the 5'-truncated fluorescent protein comprises a nucleotide sequence of SEQ ID NO: 3 or a nucleotide sequence having at least 80% homology, more concretely at least 90% homology, most concretely at least 95% homology, with SEQ ID NO: 3.
  • the 3'-truncated fluorescent protein comprises an amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 90% homology, more concretely at least 95% homology, with SEQ ID NO: 2.
  • the nucleotide encoding the 3'-truncated fluorescent protein comprises a nucleotide sequence of SEQ ID NO: 4 or a nucleotide sequence having at least 80% homology, more concretely at least 90% homology, most concretely at least 95% homology, with SEQ ID NO: 4.
  • nucleotides the variations may be purely genetic, i.e., ones that do not result in changes in the protein product. This includes nucleic acids that contain functionally equivalent codons, or codons that encode the same amino acid, such as six codons for arginine or serine, or codons that encode biologically equivalent amino acids.
  • nucleic acid molecule of this invention may encompass sequences having substantial identity to them. Sequences having the substantial identity show at least 60%, concretely at least 70%, more concretely at least 80%, even more concretely at least 90%, and most concretely at least 95% similarity to the nucleic acid molecule of this invention, as measured using one of the sequence comparison algorithms. Methods of alignment of sequences for comparison are well-known in the art.
  • the transposable element is LTR retrotransposon or proviral DNA.
  • the LTR retrotransposon or proviral DNA lacks long terminal repeat (LTR) regions. Therefore, according to the present invention, the 3'-truncated fluorescent protein is linked to the 3' end of the transposable element, the primer binding site (PBS), and the 5'-truncated fluorescent protein is linked to the 5' end of the transposable element, the polypurine tract (PPT).
  • PBS primer binding site
  • PPT polypurine tract
  • a method for detecting a transposition of a nucleic acid molecule within a genome comprising:
  • detecting has comprehensive meaning including any action of accessing information regarding the existence, level or activity of a target of interest in a sample, e.g ., transposition of a nucleic acid. Therefore, the phrase “detecting transposition” is used interchangeably with “measuring transposition”, “assessing transposition activity” or “tracing transposition”.
  • the eukaryotic cell is a plant cell.
  • plant(s) as used herein, is understood by a meaning including a plant cell, a plant tissue and a plant seed as well as a mature plant.
  • the plants applicable of the present method include, but not limited to, most dicotyledonous plants including lettuce, chinese cabbage, potato and radish, and most monocotyledonous plants including rice plant, barley and banana tree.
  • the present method can be applied to the plants selected from the group consisting of food crops such as rice plant, wheat, barley, corn, bean, potato, Indian bean, oat and Indian millet; vegetable crops such as Arabidopsis sp., Chinese cabbage, radish, red pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, welsh onion, onion and carrot; crops for special use such as ginseng, tobacco plant, cotton plant, sesame, sugar cane, sugar beet, Perilla sp., peanut and rape; fruit trees such as apple tree, pear tree, jujube tree, peach tree, kiwi fruit tree, grape tree, citrus fruit tree, persimmon tree, plum tree, apricot tree and banana tree; flower
  • the eukaryotic cell is a mammalian cell.
  • the present invention could also be applied to non-plant systems.
  • retroviruses share many features with LTR retrotransposons, and may be transformed into an LTR retrotransposon through inactivation or deletion of the domains that enable extracellular mobility. Therefore, the present invention may provide a crucial information regarding retroviral infection and transpositional issues in a host animal including a human.
  • the present invention provides a recombinant nucleic acid molecule in which a truncated fluorescent protein gene is linked to both end thereof, and a method for detecting the transposition of genetic material using the same.
  • the recombinant nucleic acid molecule of the present invention may be effectively used for tracing transposition of a gene in real-time and at single-nuclei resolution only by triggering sequential transcription and reverse transcription using a promoter-inducing chemical without awaiting an activation of transposable element mobility in following generations.
  • the method of the present invention may be applied as reliable and effective research tool for investigating genetic transpositional issue in an organism, as well as the transposition dynamics in time and space.
  • Fig. 1 shows the process of consecutive transcrtion and reverse-transcription of LTR retrotransposon (left) and the recombinant transposon of the present invention in which LTR regions have been replaced by truncated GFP gene (right).
  • Fig. 2 represents the validation of RUM methods in tobacco transient expression system.
  • Figs. 2a-2c show the overall workflow for transposition detection (Fig. 2a), GFP expressed tobacco cell (Fig. 2b) and transpositional rate of each experiments (Fig. 2c), respectively.
  • Fig. 3 represents the validation of RUM methods in stable RUM transgenic Arabidopsis systems.
  • Figs. 3a-3d show the overall workflow for transposition detection (Fig. 3a), GFP expressed nuclei counting in wild-type (Fig. 3b) and stable RUM Arabidopsis plant (Fig. 3c), and the transpositional rate of each experiments (Fig. 3d), respectively .
  • Tobacco plants were prepared for tobacco transient assay.
  • Arabidopsis Col-0 and Arabidopsis RUM plants were grown for protoplast and nuclei counting as described previously (Kim et al., 2021).
  • GFP fluorescence in transiently RUM expressed tobacco leaves was observed under Zeiss LSM880 confocal microscopy.
  • the GFP was excited at 488 nm and detected at 491-535 nm.
  • Protoplasts were isolated and counted from transiently RUM expressed tobacco leaves as described by Bargmann et al. (2010).
  • Example 1 A novel approach to study transposon mobility
  • transposon display is the most widely used experimental method to identify neo-inserted copies of a transposon.
  • a transposon display experiment must be performed in the following generations after the activation of TE mobility so that new insertions can be fixed in the genome and easily identified. This essentially limits our view of the landscape of transposition as only meiotically heritable insertions can be detected by this method.
  • RUM Real-time jUMping of transposon
  • LTR long terminal repeat
  • the original copy of artificial transposon does not produce any functional GFP signal since none of them can code for complete GFP protein.
  • the sequential reverse transcription reaction reconstitutes these incomplete GFP genes into the intact and functional fluorescence gene upon the transcriptional activation driven by the estradiol chemical-inducible promoter. Consequently, the inserted transposon DNA can produce functional GFP proteins and therefore specifically marks the cells that have undergone transposition.
  • RUM is an artificial and naive transposon, it contains the ORF of an endogenous transposon, which might induce the epigenetic silencing of the parental RUM copy.
  • RUM is an artificial and naive transposon, it contains the ORF of an endogenous transposon, which might induce the epigenetic silencing of the parental RUM copy.
  • the phenotype of RUM fluorescence is caused by the insertion of RUM DNA, which is essentially an irreversible process.

Abstract

The present invention relates to a recombinant nucleic acid molecule in which a truncated fluorescent　protein gene is linked to both end thereof, and a method for detecting the transposition of genetic material using the same. The recombinant nucleic acid molecule of the present invention may be effectively used for tracing transposition of a gene in real-time and at single-nuclei resolution only by triggering sequential transcription and reverse transcription using a promoter-inducing chemical without awaiting an activation of transposable element mobility in following generations. The method of the present invention may be applied as reliable and effective research tool for investigating genetic transpositional issue in an organism, as well as the transposition dynamics in time and space.

Description

A METHOD FOR TRACKING TRANSPOSITION OF GENETIC MATERIAL
The present invention relates to a recombinant transposable element and a method for detecting and tracking a transpositional issue in an organism using the same.
Transposable element (TE, transposon) is a stretch of DNA that can move from one position to another in the genome. Evolutionarily it has served as a major source of genetic variability, which is critical for the diversity and evolution of a species. On the other hand, transposons are also potentially dangerous because they can cause fatal mutations as they insert into genes. The importance of transposons in plant science has been growing recently as gene editing technologies are becoming more available in crop plants. The current gene editing methods exclusively require tissue culture techniques by which massive transposons can be activated, and therefore crop genome integrity can be damaged. Crop genomes are, in fact, even more vulnerable to these natural mutagens because they usually contain myriads of transposons by up to around 90%. Transposon-imposed genomic instability is a considerable bottleneck to modern crop breeding, and therefore understanding their mobility is very important. However, despite the long history of transposons, the discovery of which dates back to the early 1940s, and its ever-growing importance in crop breeding, we are still far from understanding how they proliferate in the genome. We are committed to address this issue in pursuit of unveiling their precise mechanisms for mobility. To this end, the present inventors have developed a ground-breaking technology to track transposon mobility in real-time and single-nuclei resolution. With the novel method of the present invention, the host regulators of transposon jumping are expected to be identified by coupling with the genome-wide genetic screening.
Throughout this application, various publications and patents are referred and citations are provided in parentheses. The disclosures of these publications and patents in their entities are hereby incorporated by references into this application in order to fully describe this invention and the state of the art to which this invention pertains.
Genomic instability frequently happens during tissue culture, which is a widely-used agricultural practice to propagate crop plants and modify genetic information. Such unwanted and unpredicted genomic alteration hampers eventually food safety which is an immediate threat to people's health. For this reason, the present inventors aim to understand the mechanisms of transposons, the primary agents of genomic instability, for the purpose of making good control of them. To tackle this problem, the present inventors have been working to establish a ground-breaking method to observe their action accurately and to dissect the detailed process of their mobility.
Accordingly, it is an object of this invention to provide a recombinant nucleic acid molecule to which a truncated fluorescent　protein gene is linked.
It is another object of this invention to provide a method for detecting a transposition of a nucleic acid molecule.
Other objects and advantages of the present invention will become apparent from the following detailed description together with the appended claims and drawings.
In one aspect of this invention, there is provided a recombinant nucleic acid molecule comprising:
(a) a transposable element;
(b) a nucleotide encoding a 3'-truncated fluorescent　protein linked to the 3' end of the transposable element;
(c) a nucleotide encoding a 5'-truncated fluorescent　protein linked to the 5' end of the transposable element; and
(d) a chemical-inducible regulatory sequence linked to the 5' end of the 5' truncated fluorescent　protein.
The present inventors have made intensive studies to develop a real-time method to trace a transposition of genetic material in a biological sample or an organism. As results, the present inventors have discovered that a recombinant transposable element in which LTR regions have been replaced by truncated fluorescent　protein gene may produce functional fluorescent signal only in case sequential reverse transcription reaction reconstitutes the incomplete fluorescent　protein genes into the intact one upon the transcriptional activation by chemical-inducible promoter. The novel recombinant transposable element suggested by the present inventors and tracing methods using the same efficiently overcome the limitations of the conventional method where transposon may be displayed in the following generations after the activation of transposable element mobility.
The term "nucleic acid molecule" as used herein has comprehensive meaning including DNA (gDNA and cDNA) and RNA molecule. A nucleotide, which is a basic construct unit of nucleic acid molecule, includes nucleotide analogues with modified sugar or base, as well as natural-occurring nucleotides (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584(1990)).
The term "genetic material" as used herein, refers to any material storing genetic information in the nuclei or an organism's cells. The genetic material includes DNA or RNA that is passed along from one generation to the next.
The term "biological sample" as used herein, refers to not only samples that are obtainable from an organism, but also any samples that may contain genetic material. Examples of the biological sample include human and animal blood, plant body fluids, human and animal waste, microbial culture liquid, cell culture liquid, virus culture liquid, biopsy culture liquid, soil and air, but are not limited thereto.
The term "regulatory sequence" as used herein, when used in reference to a gene or genome, refers to a DNA sequences which are necessary to control the expression of sequences to which they are ligated. The regulatory sequences may differ depending upon the intended host organism and upon the nature of the sequence to be expressed. The term "chemical-inducible regulatory sequence" refers to a regulatory sequence, e.g., a promoter, that are activated by exposure to a specific chemical compound.
According to a concrete embodiment, the chemical-inducible regulatory sequence is estradiol-inducible promoter sequence.
The term "promoter" as used herein refers to a region of DNA upstream from the transcription start and which is involved in binding RNA polymerase and other proteins to start transcription. An Estradiol-inducible promoter enables selective initiation of transcription based on the existence of beta-estradiol in biological sample.　
According to a concrete embodiment, the 3'-truncated fluorescent　protein and the 5'-truncated fluorescent　protein are each independently selected from the group consisting of green fluorescent protein (GFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), blue fluorescent protein (BFP) and yellow fluorescent protein (YFP). More concretely, the 3'-truncated fluorescent　protein and the 5'-truncated fluorescent　protein are GFP.
Since the fluorescent　protein genes linked each end of the transposable element are truncated, original copy of the recombinant transposon may not produce fluorescent signal. However, addition of a chemical, e.g., estradiol, triggers sequential reverse transcription thereby reconstitutes incomplete fluorescent　protein gene into functional one to exhibit detectable fluorescent signal.
The term "truncated" as used herein refers to a deletion of nucleotide or amino acid sequence to an extent where the biological activity of the full-length nucleotide or peptide may not be maintained. Therefore, any biologically equivalent variations are excluded from the "truncated protein" of the present invention.
According to a concrete embodiment, the 3'-truncated fluorescent　protein linked to the 3' end of the transposable element and the 5'-truncated fluorescent　protein linked to the 5' end of the transposable element share at least 30 residues of the common amino acids, and more concretely, at least 40 residues of the common amino acids.
According to a concrete embodiment, the 5'-truncated fluorescent　protein comprises an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 90% homology, more concretely at least 95% homology, with SEQ ID NO: 1.
More concretely, the nucleotide encoding the 5'-truncated fluorescent　protein comprises a nucleotide sequence of SEQ ID NO: 3 or a nucleotide sequence having at least 80% homology, more concretely at least 90% homology, most concretely at least 95% homology, with SEQ ID NO: 3.
According to a concrete embodiment, the 3'-truncated fluorescent　protein comprises an amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 90% homology, more concretely at least 95% homology, with SEQ ID NO: 2.
More concretely, the nucleotide encoding the 3'-truncated fluorescent　protein comprises a nucleotide sequence of SEQ ID NO: 4 or a nucleotide sequence having at least 80% homology, more concretely at least 90% homology, most concretely at least 95% homology, with SEQ ID NO: 4.
It would be obvious to the skilled artisan that the nucleotide sequences used in this invention are not limited to those listed in the appended Sequence Listings.
For nucleotides, the variations may be purely genetic, i.e., ones that do not result in changes in the protein product. This includes nucleic acids that contain functionally equivalent codons, or codons that encode the same amino acid, such as six codons for arginine or serine, or codons that encode biologically equivalent amino acids.
Considering biologically equivalent variations described hereinabove, the nucleic acid molecule of this invention may encompass sequences having substantial identity to them. Sequences having the substantial identity show at least 60%, concretely at least 70%, more concretely at least 80%, even more concretely at least 90%, and most concretely at least 95% similarity to the nucleic acid molecule of this invention, as measured using one of the sequence comparison algorithms. Methods of alignment of sequences for comparison are well-known in the art.
According to a concrete embodiment, the transposable element is LTR retrotransposon or proviral DNA.
More concretely, the LTR retrotransposon or proviral DNA lacks long terminal repeat (LTR) regions. Therefore, according to the present invention, the 3'-truncated fluorescent　protein is linked to the 3' end of the transposable element, the primer binding site (PBS), and the 5'-truncated fluorescent　protein is linked to the 5' end of the transposable element, the polypurine tract (PPT).
In another aspect of this invention, there is provided a method for detecting a transposition of a nucleic acid molecule within a　genome comprising:
(a) preparing a recombinant nucleic acid molecule of the present invention above mentioned;
(b) introducing the nucleic acid molecule to an eukaryotic cell;
(c) treating the cell with a chemical which activates the regulatory sequence.
(d) measuring a fluorescence exhibited by the cell.
As the recombinant nucleic acid molecules of the present are mentioned above, they are omitted herein to avoid excessive overlaps.
The term "detecting" as used herein, has comprehensive meaning including any action of accessing information regarding the existence, level or activity of a target of interest in a sample, e.g., transposition of a nucleic acid. Therefore, the phrase "detecting transposition" is used interchangeably with "measuring transposition", "assessing transposition activity" or "tracing transposition".
According to a concrete embodiment, the eukaryotic cell is a plant cell.
The term "plant(s)" as used herein, is understood by a meaning including a plant cell, a plant tissue and a plant seed as well as a mature plant.
The plants applicable of the present method include, but not limited to, most dicotyledonous plants including lettuce, chinese cabbage, potato and radish, and most monocotyledonous plants including rice plant, barley and banana tree. Preferably, the present method can be applied to the plants selected from the group consisting of food crops such as rice plant, wheat, barley, corn, bean, potato, Indian bean, oat and Indian millet; vegetable crops such as Arabidopsis sp., Chinese cabbage, radish, red pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, welsh onion, onion and carrot; crops for special use such as ginseng, tobacco plant, cotton plant, sesame, sugar cane, sugar beet, Perilla sp., peanut and rape; fruit trees such as apple tree, pear tree, jujube tree, peach tree, kiwi fruit tree, grape tree, citrus fruit tree, persimmon tree, plum tree, apricot tree and banana tree; flowering crops such as rose, gladiolus, gerbera, carnation, chrysanthemum, lily and tulip; and fodder crops such as ryegrass, red clover, orchardgrass, alfalfa, tallfescue and perennial ryograss.
According to a concrete embodiment, the eukaryotic cell is a mammalian cell.
The present invention could also be applied to non-plant systems. For example, numerous studies in various eukaryotes, including mammals, found that retrotransposons are transcriptionally activated by certain diseases or at particular stages. It was also suggested that transposition might be an important component of disease progression.
Particularly, retroviruses share many features with LTR retrotransposons, and may be transformed into an LTR retrotransposon through inactivation or deletion of the domains that enable extracellular mobility. Therefore, the present invention may provide a crucial information regarding retroviral infection and transpositional issues in a host animal including a human.
Throughout this specification and the claims, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated member, integer or step or group of members, integers or steps but not the exclusion of any other member, integer or step or group of members, integers or steps. The terms "a" and "an" and "the" and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as", "for example"), provided herein is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
The features and advantages of the present invention will be summarized as follows:
(a) The present invention provides a recombinant nucleic acid molecule in which a truncated fluorescent　protein gene is linked to both end thereof, and a method for detecting the transposition of genetic material using the same.
(b) The recombinant nucleic acid molecule of the present invention may be effectively used for tracing transposition of a gene in real-time and at single-nuclei resolution only by triggering sequential transcription and reverse transcription using a promoter-inducing chemical without awaiting an activation of transposable element mobility in following generations.
(c) The method of the present invention may be applied as reliable and effective research tool for investigating genetic transpositional issue in an organism, as well as the transposition dynamics in time and space.
Fig. 1 shows the process of consecutive transcrtion and reverse-transcription of LTR retrotransposon (left) and the recombinant transposon of the present invention in which LTR regions have been replaced by truncated GFP gene (right).
Fig. 2 represents the validation of RUM methods in tobacco transient expression system. Figs. 2a-2c show the overall workflow for transposition detection (Fig. 2a), GFP expressed tobacco cell (Fig. 2b) and transpositional rate of each experiments (Fig. 2c), respectively.
Fig. 3 represents the validation of RUM methods in stable RUM transgenic Arabidopsis systems. Figs. 3a-3d show the overall workflow for transposition detection (Fig. 3a), GFP expressed nuclei counting in wild-type (Fig. 3b) and stable RUM Arabidopsis plant (Fig. 3c), and the transpositional rate of each experiments (Fig. 3d), respectively .
The present invention will now be described in further detail by examples. It would be obvious to those skilled in the art that these examples are intended to be more concretely illustrative and the scope of the present invention as set forth in the appended claims is not limited to or by the examples.
EXAMPLES
The present invention will now be described in further detail by examples. It would be obvious to those skilled in the art that these examples are intended to be more concretely illustrative and the scope of the present invention as set forth in the appended claims is not limited to or by the examples.
EXAMPLES
MATERIALS AND METHODS
1. Plant materials and growth condition
Tobacco plants were prepared for tobacco transient assay. Arabidopsis Col-0 and Arabidopsis RUM plants were grown for protoplast and nuclei counting as described previously (Kim et al., 2021).
2. Microscopy analysis
GFP fluorescence in transiently RUM expressed tobacco leaves was observed under Zeiss LSM880 confocal microscopy. The GFP was excited at 488 nm and detected at 491-535 nm.
3. Protoplast isolation and counting
Protoplasts were isolated and counted from transiently RUM expressed tobacco leaves as described by Bargmann et al. (2010).
4. Nuclei isolation and counting
Nuclei were isolated and counted from b-estradiol treated Arabidopsis Col-0 and Arabidopsis RUM transgenic plants as described previously by Moro et al. (2021).
Example 1: A novel approach to study transposon mobility
The activity of transposons has been mostly investigated at transcriptional and post-transcriptional steps; however, the transpositional behavior of transposons has been technically challenging to study. It is quite difficult to be discriminated from the parental copies because of a newly inserted copy. The complex mosaic pattern of somatic transposition makes it even more difficult to identify new insertions of transposons. For example, transposon display is the most widely used experimental method to identify neo-inserted copies of a transposon. However, a transposon display experiment must be performed in the following generations after the activation of TE mobility so that new insertions can be fixed in the genome and easily identified. This essentially limits our view of the landscape of transposition as only meiotically heritable insertions can be detected by this method.
To overcome this drawback, we developed a unique and novel technique that allows us to trace the transposition in real-time and at single-nuclei resolution (tentatively termed "RUM" for Real-time jUMping of transposon). As illustrated below, RUM employs two truncated GFP genes placed at the long terminal repeat (LTR) regions. The original copy of artificial transposon does not produce any functional GFP signal since none of them can code for complete GFP protein. The sequential reverse transcription reaction reconstitutes these incomplete GFP genes into the intact and functional fluorescence gene upon the transcriptional activation driven by the estradiol chemical-inducible promoter. Consequently, the inserted transposon DNA can produce functional GFP proteins and therefore specifically marks the cells that have undergone transposition.
So far, we have tested the RUM method in both tobacco transient expression and stable transgenic Arabidopsis systems. Consistently, we were able to measure for the first time ever in plant systems the accurate transposition rate detected at around 1%. Worth noting is that the Fluorescence-activated Cell Sorting (FACS) equipment allows us not only the high-throughput and rapid detection of transposition rate, but also efficient collection of nuclei tagged with new-insertion of RUM. Apparently, RUM is a ground-breaking technique that enables detection of TE mobility at unprecedented precision and resolution.
Example 2 : Identification of the transposition regulators
We are eager to address the fundamental question using the RUM approach is how the host genome controls the transposition. We plan to address this question by identifying the trans-acting host regulators of TE mobility. In designing a forward genetic screening, we had to consider several critical points of using the RUM system. Firstly, although RUM is an artificial and naive transposon, it contains the ORF of an endogenous transposon, which might induce the epigenetic silencing of the parental RUM copy. Secondly, the phenotype of RUM fluorescence is caused by the insertion of RUM DNA, which is essentially an irreversible process. These unusual and complex features of the RUM assay made it impossible to carry out the classical forward genetic screening approach.
Being aware of the limitations of the conventional genetic approach and the uniqueness of the RUM system, we devised an entirely new genetic screening approach that will allow us to identify the candidate genes involved in the transposition process. Taking advantage of the high-throughput synthesis system of oligonucleotides and CRISPR-Cas9 mutagenesis approach, we generated a genome-wide CRISPR library that covers all protein-coding genes of Arabidopsis in average by three independent guide RNAs. By performing a large-scale Agrobacterium-mediated plant transformation of this CRISPR library, we obtained a pool of transgenic plants, each of which in principle contains a single guide RNA targeting a specific gene. We will then carry out the RUM assay using a large pool of plants that will be followed by a collection of GFP-positive nuclei, which will be sequenced in NGS platforms. The difference of our approach from classical genetic screening is that we only determine the enrichment of guide RNA reads from NGS data which would indicate the strength of influence by individual guide RNA. It is noteworthy that our genome-wide CRISPR mutagenesis approach is time-efficient because we will be able to identify candidate genes in only one generation.
Example 3 : Potential pitfalls and strategy
We have carefully considered possible pitfalls that may arise. An issue that requires a special note here is that the regular selection methods of transgenic plants using antibiotics can be a considerable disadvantage in our approach for several reasons. First, RUM activation requires the treatment of an estradiol chemical and additional treatment of chemical reagents may reduce plant fitness. Second, the antibiotics selection of transgenic plants is not very efficient for high-throughput experiments. To bypass such issues, we engineered our CRISPR-Cas9 vector to replace the antibiotics resistance gene with the seed coat fluorescence gene. The fluorescence signal from the seed coat enables the selection of transgenic seeds even prior to germination, which allows us essentially to grow only the transformed plants on media. With this brilliant amendment of the selection method, we were able to increase the throughput and efficiency of our assay system even higher.
Having described a preferred embodiment of the present invention, it is to be understood that variants and modifications thereof falling within the spirit of the invention may become apparent to those skilled in this art, and the scope of this invention is to be determined by appended claims and their equivalents.
References
1. Bargmann B.O.R., Birnbaum K.D., Fluorescence Activated Cell Sorting of Plant Protoplasts. Jove, doi: 10.3791/1673 (2010).
2. Kim E.Y., Wang L., Lei Z., Li H., Fan W., Cho J., Ribosome stalling and SGS3 phase separation prime the epigenetic silencing of transposons. Nature Plants, 7: 303-309 (2021).
3. Moro B., Kisielow M., Borrero V.B., Bouet A., Brosnan C.A., Bologna N.G., Nuclear RNA purification by flow cytometry to study nuclear processes in plants. STAR Protocols, 2: 1000320 (2021).

Claims (13)

  1. A recombinant nucleic acid molecule comprising:
    (a) a transposable element;
    (b) a nucleotide encoding a 3'-truncated fluorescent　protein linked to the 3' end of the transposable element;
    (c) a nucleotide encoding a 5'-truncated fluorescent　protein linked to the 5' end of the transposable element; and
    (d) a chemical-inducible regulatory sequence linked to the 5' end of the 5' truncated fluorescent　protein.
  2. The nucleic acid molecule according to claim 1, wherein the 3'-truncated fluorescent　protein and the 5'-truncated fluorescent　protein are each independently selected from the group consisting of green fluorescent protein (GFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), blue fluorescent protein (BFP) and yellow fluorescent protein (YFP).
  3. The nucleic acid molecule according to claim 2, wherein the 3'-truncated fluorescent　protein and the 5'-truncated fluorescent　protein are GFP.
  4. The nucleic acid molecule according to claim 3, wherein the 5'-truncated fluorescent　protein comprises an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 90% homology with SEQ ID NO: 1.
  5. The nucleic acid molecule according to claim 4, wherein the nucleotide encoding the 5'-truncated fluorescent　protein comprises a nucleotide sequence of SEQ ID NO: 3 or a nucleotide sequence having at least 80% homology with SEQ ID NO: 3.
  6. The nucleic acid molecule according to claim 3, wherein the 3'-truncated fluorescent　protein comprises an amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 90% homology with SEQ ID NO: 2.
  7. The nucleic acid molecule according to claim 6, wherein the nucleotide encoding the 3'-truncated fluorescent　protein comprises a nucleotide sequence of SEQ ID NO: 4 or a nucleotide sequence having at least 80% homology with SEQ ID NO: 4.
  8. The nucleic acid molecule according to claim 1, wherein the chemical-inducible regulatory sequence is estradiol-inducible promoter sequence.
  9. The nucleic acid molecule according to claim 1, wherein the transposable element is LTR retrotransposon or proviral DNA.
  10. The nucleic acid molecule according to claim 8, wherein the LTR retrotransposon or proviral DNA lacks long terminal repeat (LTR) regions.
  11. A method for detecting a transposition of a nucleic acid molecule within a　genome comprising:
    (a) preparing a recombinant nucleic acid molecule according to any one of claims 1 to 10;
    (b) introducing the nucleic acid molecule to an eukaryotic cell;
    (c) treating the cell with a chemical which activates the regulatory sequence.
    (d) measuring a fluorescence exhibited by the cell.
  12. The method according to claim 11, wherein the eukaryotic cell is a plant cell.
  13. The method according to claim 11, wherein the eukaryotic cell is a mammalian cell.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070294788A1 (en) * 2002-05-01 2007-12-20 University Of Georgia Research Foundation Transposable Elements in Rice and Methods of Use
WO2020230909A1 (en) * 2019-05-10 2020-11-19 Cas Center For Excellence In Molecular Plant Sciences A method for detecting transposable element in a biological sample

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070294788A1 (en) * 2002-05-01 2007-12-20 University Of Georgia Research Foundation Transposable Elements in Rice and Methods of Use
WO2020230909A1 (en) * 2019-05-10 2020-11-19 Cas Center For Excellence In Molecular Plant Sciences A method for detecting transposable element in a biological sample

Non-Patent Citations (3)

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Title
BÖHMDORFER GUDRUN, TRAMONTANO ANDREA, LUXA KERSTIN, BACHMAIR ANDREAS: "A synthetic biology approach allows inducible retrotransposition in whole plants", SYSTEMS AND SYNTHETIC BIOLOGY, vol. 4, no. 2, 1 June 2010 (2010-06-01), NL , pages 133 - 138, XP093023627, ISSN: 1872-5325, DOI: 10.1007/s11693-010-9053-4 *
DATABASE Protein 11 October 2006 (2006-10-11), ANONYMOUS: "(GAGP)3-EGFP fusion protein [synthetic construct]", XP093023623, retrieved from Genbank Database accession no. ABE28521 *
DATABASE Protein 6 April 2020 (2020-04-06), ANONYMOUS: "enhanced green fluorescent protein (plasmid) [Bacillus cereus]", XP093023626, retrieved from Genbank Database accession no. ACV20892 *

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