JP7766300B2 - Method for modifying the methylation status of genomic DNA - Google Patents

Description

The present invention relates to a method for modifying the methylation status of genomic DNA and a method for suppressing gene expression using single-stranded DNA.

A technique for long-term suppression of specific gene expression, called RNAi, is used in many species, both animals and plants, using genetic engineering to incorporate a cassette that transcribes shRNA (short hairpin RNA) into the genome (Non-Patent Document 1). Another method used exclusively in land plants has been reported to suppress target gene expression using the RdDM (RNA-directed DNA methylation) mechanism (Non-Patent Document 2). Furthermore, for cultured animal cells, methods have been reported in which protein fragments that recognize specific base sequences, such as TALE, ZnF, and dCas9, are fused with enzymes that catalyze DNA methylation or histone demethylation, to control the expression of specific genes (Non-Patent Documents 3-5).

However, these gene expression control techniques using genetic recombination and genome editing require a relatively long time to obtain the desired strain. Furthermore, when using base sequence recognition proteins for genome editing, their creation is an extremely complicated process, and there is also the issue that compatibility between the protein and the host can affect the efficiency of genome editing.

Paddison P. J. et al., Genes Dev. 2002, 16, 948-958 Marjori A. Matzke et al., Nature Reviews Genetics, 2014, 15, 394-408 Morgan L. Maeder et al., Nature Biotechnology, 2013, 31(12), 1137-1142 Hui Chen et al., Nucleic Acids Research, 2014, 42(3), 1563-1574 Angelo Amabile et al., cell, 2016, 167(1), 219-232

The present invention was made in light of these circumstances, and its object is to provide a method for simply and efficiently modifying the methylation status of genomic DNA. A further object of the present invention is to provide a method for simply and efficiently suppressing the expression of a specific gene through modification of the methylation status.

As a result of intensive research aimed at solving the above-mentioned problems, the inventors discovered that it is possible to modify the methylation state of genomic DNA in a target DNA region simply by introducing into cells single-stranded DNA having a base sequence homologous to that of the target DNA region. Furthermore, the inventors discovered that by introducing into cells single-stranded DNA having a base sequence homologous to the base sequence of the sense strand of a specific gene, it is possible to modify the methylation state around the gene region and specifically and significantly suppress the expression of the gene over a long period of time, leading to the completion of the present invention.

More specifically, the present invention provides the following aspects:

(1) A method for altering the methylation status of genomic DNA, characterized by introducing into a cell single-stranded DNA having a base sequence homologous to the base sequence of the DNA region in the genomic DNA whose methylation status is desired to be altered.

(2) A method for suppressing the expression of a gene in genomic DNA, characterized by introducing into a cell single-stranded DNA having a base sequence homologous to the base sequence of the sense strand of the gene whose expression is to be suppressed in the genomic DNA.

(3) A method described in (1) or (2), wherein the single-stranded DNA is 50 to 120 bases long.

(4) A method described in any one of (1) to (3), wherein the cells are algae.

(5) A composition or kit for producing cells in which the methylation status of genomic DNA has been altered, comprising single-stranded DNA having a base sequence homologous to the base sequence of a DNA region in which the methylation status of the genomic DNA is desired to be altered.

(6) A composition or kit for producing cells in which the expression of a gene in genomic DNA is suppressed, comprising single-stranded DNA having a base sequence homologous to the base sequence of the sense strand of the gene whose expression is to be suppressed in the genomic DNA.

According to the present invention, the simple procedure of introducing single-stranded DNA of a certain length into cells makes it possible to modify the methylation status of bases in target DNA regions, thereby efficiently suppressing the expression of specific genes. Because the method of the present invention does not rely on a specific polymerase, as in RdDM-dependent epigenetic editing, it can be applied to a wide range of biological species. Furthermore, compared to methods that use base-recognition proteins, it is possible to modify the methylation status of multiple bases over a wide range at once.

Figure 1 shows the DNA regions from which single-stranded DNAs were designed. Single-stranded DNAs were designed based on the base sequences of two sites in the 5' untranslated region of the GPF gene (Prom1 and Prom2; Scheme A) and four sites within the ORF (ssDNA1, ssDNA2, ssDNA3, and ssDNA4; Scheme B). 1 is a graph showing the efficiency of suppressing GFP expression in haptophytes into which ssDNA1 or ssDNA2 has been introduced. Electrophoresis photograph (left) and graph (right) showing the results of expression analysis of the GFP gene in haptophytes whose fluorescence was abolished by the introduction of ssDNA1 or ssDNA2. This figure shows the results of analyzing the methylation status in the region (region 1) containing the promoter and the 5' end of the open reading frame (ORF) in haptophyte algae whose fluorescence was abolished by the introduction of ssDNA1 or ssDNA2. Region 1 contains 80 cytosine bases, and bases that showed a change in methylation status compared to the wild type (WT) were selected. The numbers in the boxes indicate the percentage of methylated cytosines. Percentage of methylated cytosines: 0-35%: white, 35-70%: gray, 70-100%: black. This figure shows the results of analyzing the methylation status in the ORF region (region 2) of engineered single-stranded DNA in haptophyte algae whose fluorescence was abolished by the introduction of ssDNA1 or ssDNA2. Region 2 contains 91 cytosine bases, and bases that showed changes in methylation status compared to the wild type (WT) were selected. The numbers in the boxes indicate the percentage of methylated cytosines. Percentage of methylated cytosines: 0-35%: white, 35-70%: gray, 70-100%: black. 1 is a graph showing the results of an analysis of the correlation between the amount of ssDNA2 introduced and the expression suppression efficiency. 1 is a graph showing the results of an analysis of the correlation between the chain length of ssDNA2 and expression suppression efficiency. 1 is a graph showing the results of an analysis of the correlation between the region for designing single-stranded DNA and expression suppression efficiency. Electrophoresis photographs showing the results of analyzing the suppression of expression of the endogenous FCP gene by single-stranded DNA. In the figure, "WT" indicates the wild type, and "B4" indicates the FCP expression suppression strain. 8B is a graph showing the results of measuring the fluorescence of the gel electrophoresed in Fig. 8A. In the figure, "WT" indicates the wild type, and "B4" indicates the FCP expression suppression strain.

The present invention provides a method for altering the methylation status of genomic DNA.

The method of the present invention is characterized by introducing into a cell single-stranded DNA having a base sequence homologous to the base sequence of a DNA region (target genomic DNA region) in which the methylation status of the genomic DNA is desired to be altered.

"Methylation of genomic DNA" is a chemical reaction in which a methyl group is added to the carbon atom of a base in genomic DNA. In the present invention, this primarily refers to the addition of a methyl group to the 5-carbon atom of the pyrimidine ring of cytosine. "Alteration of the methylation state" in the present invention includes both an increase and a decrease (demethylation) in genomic DNA methylation, but primarily refers to an increase in methylation. The relationship between genomic DNA methylation and the suppression of gene expression is known, and as described below, the expression of specific genes can be suppressed by increasing methylation of genomic DNA using the methods of the present invention.

Changes in the methylation status of genomic DNA are known to occur in both eukaryotic and prokaryotic cells, and there are no particular limitations on the "cells" whose methylation status of genomic DNA is modified in the methods of the present invention. The cells may be eukaryotic cells such as algal cells, plant cells, animal cells, and fungal cells, or prokaryotic cells such as bacteria and archaea.

Algae include eukaryotic algae such as haptophytes, cryptophytes, brown algae, red algae, green algae, diatoms, golden algae, glaucophytes, euglenids, charophytes, and dinoflagellates, as well as prokaryotic algae such as cyanobacteria. Plants include seed plants, ferns, and bryophytes. Examples of experimentally or industrially useful plants include Arabidopsis thaliana, tomato, soybean, rice, wheat, barley, corn, rapeseed, tobacco, banana, peanut, sunflower, potato, cotton, and carnation. Animals include, for example, mammals (e.g., mice, rats, guinea pigs, hamsters, rabbits, humans, monkeys, pigs, cows, goats, and sheep), as well as fish, birds, reptiles, amphibians, and insects.

"Cells" includes cultured cells as well as cells in an individual. It also includes cells, such as protoplasts, that have been specially treated to introduce single-stranded DNA.

In the method of the present invention, the "single-stranded DNA" introduced into cells has a base sequence homologous to the base sequence of the target strand of the DNA region in genomic DNA where it is desired to modify the methylation state (target genomic DNA region). According to the present invention, any genomic DNA region can be targeted for modification of the methylation state. Furthermore, the "target strand" is the strand of double-stranded genomic DNA that has a base sequence homologous to the single-stranded DNA of the present invention and serves as the basis for designing the single-stranded DNA of the present invention. For example, when methylating a gene region using the single-stranded DNA of the present invention, the target strand is the sense strand. Here, "sense strand" refers to the strand of double-stranded genomic DNA that does not serve as a transcription template.

The length of the single-stranded DNA is not particularly limited as long as it can modify the methylation state, but is, for example, 40 to 200 bases, preferably 50 to 120 bases.

The homology between the single-stranded DNA and the target genomic DNA region does not necessarily have to be 100%, as long as it is possible to modify the methylation status of the genomic DNA. The homology can be, for example, 90% or more (e.g., 95% or more, 96% or more, 97% or more, 98% or more, 99% or more). Sequence homology can be calculated using BLAST or similar tools (e.g., default parameters).

Methods for introducing single-stranded DNA into cells can be selected appropriately depending on the type and morphology of the cells. Examples include, but are not limited to, electroporation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, lipofection, nanoparticle-mediated transfection, and virus-mediated nucleic acid delivery.

The concentration of single-stranded DNA to be introduced into cells is not limited as long as it can modify the methylation state of genomic DNA, and can be appropriately determined by those skilled in the art depending on the length of the single-stranded DNA and the type of cells to be introduced. For example, the concentration is 1 ng to 50 μg per ¹⁰ cells, and preferably 6 ng to 8 μg per ¹⁰ cells.

Changes in methylation status due to the methods of the present invention can be analyzed using known methods such as bisulfite sequencing, methylation-specific PCR (MSP), quantitative MSP, COBRA, and pyrosequencing, and differences in methylation status compared to a control (e.g., when single-stranded DNA is not introduced) can be evaluated as alterations in methylation status.

The method of the present invention alters the methylation state not only at the bases in genomic DNA for which single-stranded DNA has been designed, but also in their vicinity. Here, "vicinity" refers to the region within 300 bases upstream from the 5' end of the base sequence for which single-stranded DNA has been designed, and the region within 300 bases downstream from the 3' end of the base sequence for which single-stranded DNA has been designed. However, this does not exclude alterations in the methylation state of bases further away from the base sequence for which single-stranded DNA has been designed.

The method of the present invention allows for the suppression of gene expression by altering the methylation state of the gene region. Therefore, the present invention also provides a method for suppressing gene expression in genomic DNA, characterized by introducing into cells single-stranded DNA having a base sequence homologous to the base sequence of the sense strand of the gene (target gene) whose expression is to be suppressed in the genomic DNA. When the purpose is to suppress gene expression, the region for which the single-stranded DNA is designed is preferably the open reading frame region of the gene. The method of the present invention makes it possible to specifically and significantly suppress gene expression over a long period of time.

The present invention also provides a composition or kit for producing cells in which the methylation status of genomic DNA has been altered, or for producing cells in which gene expression in genomic DNA has been suppressed, comprising the above-mentioned single-stranded DNA.

The preparations constituting the kits of the present invention and the compositions of the present invention may further contain other components as necessary. Examples of other components include, but are not limited to, bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, chelating agents, etc.

The kit of the present invention may further comprise additional components. Examples of additional components include, but are not limited to, a dilution buffer, a wash buffer, a nucleic acid transfer reagent, and a control reagent (e.g., control single-stranded DNA). The kit may also include instructions for carrying out the method of the present invention.

Materials and Methods
(1) Experimental species
In experiments on GFP (Green Fluorescence Protein) expression suppression, the SG-2 strain (a strain that constitutively expresses GFP) was used as the experimental subject, in which the GFP gene and the Aph7 gene (a hygromycin resistance gene) were introduced into the haptophyte algae (Pleurochrysis carterae LU strain). In experiments on FCP (Fucoxanthin chlorophyll a/c-binding protein), a wild-type strain of the haptophyte algae (Pleurochrysis carterae LU strain) was used. Cells were cultured in Marine Art-ESM medium at 20°C under a 16-hour light/8-hour dark cycle.

(2) Protoplast Preparation and Introduction of Single-Stranded DNA. Cells were collected from late stationary phase cell cultures by centrifugation and treated with Protanase K at 30°C for 3 hours (shaking) and at 20°C for 1 hour (static). The collected cells were treated in hypo-osmotic buffer for 5–10 minutes to remove the cell wall. The resulting protoplasts were filtered through Miracloth (EMD Millipore), collected by centrifugation, and suspended in 0.4 M mannitol solution. The suspension was centrifuged again, the supernatant discarded, and the cells were resuspended in 0.4 M mannitol solution and counted. The protoplast suspension was centrifuged again, and 4.2 × 10 ⁵ cells were suspended in 320 μL of MaMg buffer and mixed with 30 μg of single-stranded DNA dissolved in 300 μL of Millipore water.

The single-stranded DNA used in the GFP expression suppression experiment was 100 bases long and had the same sequence as the sense strand of two regions within the ORF of the GFP gene (hereafter referred to as "ssDNA1" and "ssDNA2"). The single-stranded DNA used in the FCP expression suppression experiment was also 100 bases long and had the same sequence as the sense strand of a portion of the ORF of the FCP gene.

The protoplast suspension containing single-stranded DNA was left to stand in the dark for 10 minutes, after which 350 μL of 40% PEG (MW = 6,000, WAKO) CMS solution was poured into it, and after thorough mixing, it was left to stand in the light at 20°C for 15 minutes. The protoplast suspension thus treated was washed with 7.5 mL of medium, suspended in 2 mL of medium, and cultured.

The composition of the solution used is as follows:

・hypo-osmotic buffer (HEPES 10mM, KCl 100mM, NaOH 350μM)
- MaMg buffer (mannitol 400mM, _MgCl2 15mM, MES 0.1% (w/v), pH adjusted to 5.8 with KOH)
・40% PEG in CMS solution (PEG 4g, CMS solution 6mL)
- CMS solution (mannitol 400 mM, Ca(NO ₃ ) ₂ 100 mM, pH adjusted to 7.0 with KOH)
(3) Calculation of GFP fluorescence expression suppression efficiency
Approximately one week after transfection of ssDNA1 or ssDNA2, the percentage of GFP-fluorescent cells and non-fluorescent cells was measured using flow cytometry. A control group without transfection of single-stranded DNA was also measured at the same time. The expression suppression efficiency was defined by the following formula:
100 - [(number of fluorescent cells in the single-stranded DNA transfection experiment group / number of fluorescent cells in the control experiment group) x 100] (%)
In this formula, for example, if the expression suppression efficiency is 80%, then fluorescence will disappear in 80% of the cells.

(4) Isolation of cells with long-term suppression of expression. The cells obtained in (2) were cultured in liquid medium for approximately 2 weeks, followed by culture in half-strength MA-ESM plate medium containing 0.2% gellan gum. After 2 weeks, colonies formed on the plates were observed under a fluorescent stereomicroscope. Colonies in which GFP fluorescence had completely disappeared were isolated and cultured again in liquid medium for approximately 4 weeks. After confirming the disappearance of fluorescence using flow cytometry, one colony each from the ssDNA1-transfected and ssDNA2-transfected experimental groups (referred to as "ssD1" and "ssD2," respectively) was selected, and RNA and genomic DNA were extracted.

(5) GFP mRNA expression analysis: Using the RNA extracted in (4), expression analysis was performed using GFP-specific primers. As a positive control, the expression of the housekeeping gene FCP was also confirmed.

(6) Analysis of genomic methylation. The genomic DNA obtained in (3) was treated with bisulfite using the Fast Bisulfite Conversion Kit (Abcam). Bisulfite treatment deaminates unmethylated C (cytosine base) and converts it to U (uracil base). By performing PCR after this treatment, unmethylated C is replaced by T (thymine base), allowing the detection of methylated and unmethylated C.

Using bisulfite-treated genomic DNA as a template, the region containing the GFP promoter region and ORF (region 1) and the ORF region (region 2) containing the introduced single-stranded DNA were amplified by PCR, TA cloning was performed, and the amplified region was introduced into E. coli (JM109 strain). Additionally, using non-bisulfite-treated genomic DNA as a template, regions 1 and 2 were PCR-cloned and cloned to serve as control sequences. Twelve E. coli clones were isolated from each experimental group and cultured, and the PCR-amplified fragments were sequenced to calculate the methylation rate.

(7) Consideration of the single-stranded DNA to be introduced
For the single-stranded DNA of GFP, the following conditions were examined.

(a) Amount of single-stranded DNA introduced In the procedure described in (2) above, ssDNA2 was introduced at amounts of 5 ng, 10 ng, 25 ng, 50 ng, 75 ng, 100 ng, 125 ng, 500 ng, 1 μg, and 1.5 μg per 4.2 ^{× 10} cells, and the expression suppression efficiency was calculated using the procedure described in (3) above.

(b) Chain length of single-stranded DNA In the procedure described in (2) above, ssDNA2 with chain lengths of 40 bases, 50 bases, 60 bases, 80 bases, and 100 bases was prepared and introduced in amounts adjusted to 4 ng, 5 ng, 6 ng, 8 ng, and 10 ng per 4.2 × ¹⁰ cells, respectively, and the expression suppression efficiency was calculated using the procedure described in (3) above.

(c) Region for designing single-stranded DNA
Single-stranded DNA consisting of 80 bases was designed at two locations in the 5' untranslated region of the GPF gene (Prom1 and Prom2; Schematic Diagram A) and two locations within the ORF (near the center and near the 3'end; ssDNA3 and ssDNA4; Schematic Diagram B) (Figure 1). 10 μg of DNA was introduced into 4.2 × ¹⁰ cells using the procedure described in (2), and the expression suppression efficiency was calculated using the procedure described in (3).

(8) Application experiments using the endogenous gene FCP (Fucoxanthin chlorophyll a/c-binding protein)
FCP is a protein that binds to photosynthetic pigments to form photoreceptors. When expression of this gene is suppressed, the receptors shrink and the cells become paler. In P. carterae, this gene forms a family of seven different sequences. Based on the sequence with the highest expression level, an 80-base single-stranded DNA was synthesized and introduced into the cells. After cultivation, cells that had turned paler were isolated and re-cultured, and the expression of the FCP gene was analyzed.

[result]
(1) Suppression of GFP expression by introduction of ssDNA1 or ssDNA2 As shown in Figure 2, the GFP expression suppression efficiency of ssDNA1 and ssDNA2 was approximately 83.8% and 87.5%, respectively.

(2) Suppression of GFP expression by introduction of ssDNA1 or ssDNA2 As shown in Figure 3, GFP gene expression was analyzed for the isolated fluorescence-quenching mutants. The results showed that GFP gene expression was suppressed to less than 1% compared to the control in both ssDNA1 and ssDNA2-transfected mutants.

(3) Analysis of methylation status. The methylation status of the two isolated strains described above was examined for the region containing the promoter and the 5' end of the ORF (region 1) and the region of the ORF containing the introduced single-stranded DNA (region 2). As shown in Figure 4, both methylation and demethylation were confirmed in the isolated strains, but newly methylated sites were more prevalent. Furthermore, a more significant difference was observed in region 2 compared to region 1.

(4) Amount of single-stranded DNA introduced and expression suppression efficiency
Using ssDNA2, we investigated the correlation between the amount of DNA introduced and the efficiency of expression suppression. As shown in Figure 5, the suppression efficiency was generally over 70% at doses of 25 ng to 30 μg per 4.2 × ¹⁰ cells, but the suppression efficiency was significantly lower in the 10 ng and 5 ng experimental groups.

(5) Relationship between single-stranded DNA length and expression suppression efficiency
We designed single-stranded DNAs of 40, 50, 60, and 80 bases in the ssDNA2 region and investigated the correlation between the length of the introduced single-stranded DNA and the expression suppression efficiency. As shown in Figure 6, the efficiency was significantly reduced when a single-stranded DNA of 40 bases in length was used.

(6) Relationship between single-stranded DNA design region and expression suppression efficiency. Single-stranded DNA was designed into several regions of the ORF and promoter, and the correlation with expression suppression efficiency was examined. As shown in Figure 7, all single-stranded DNA designed within the ORF showed a suppression efficiency of 75% or more, but the suppression efficiency of DNA designed into the promoter region was significantly lower (approximately 5%).

(7) Gene expression suppression experiment using the endogenous gene FCP
After transfection with single-stranded FCP DNA, colonies that had become pale were isolated and subjected to FCP gene expression analysis. As shown in Figure 8, expression of all seven subtypes except type 5 was suppressed.

The present invention makes it possible to modify the functions of various cells and individuals by altering the methylation state of genomic DNA and suppressing the expression of specific genes. Therefore, the present invention is expected to be useful in a wide range of fields, including industry, agriculture, and medicine. For example, by applying the present invention to haptophyte microalgae, it is possible to efficiently create high-lipid-producing strains and strains that highly produce useful lipids. By using the present invention, cells and individuals with modified functions can be obtained without genetic modification. Therefore, compared to genetically modified individuals, the present invention is advantageous for industrial application in that there are fewer restrictions on facilities, etc., imposed by regulations (e.g., the Cartagena Protocol).

Claims (6)

A method for modifying the methylation status of genomic DNA, characterized by introducing single-stranded DNA having a base sequence homologous to the base sequence of the DNA region in the genomic DNA whose methylation status is to be modified into cells (excluding cells in human individuals) without using DNA methyltransferase. A method for suppressing gene expression in genomic DNA, characterized by introducing single-stranded DNA having a base sequence homologous to the base sequence of the sense strand of the gene whose expression is to be suppressed in genomic DNA into cells (excluding cells in human individuals) without using DNA methyltransferase. The method of claim 1 or 2, wherein the single-stranded DNA is 50 to 120 bases long. The method of any one of claims 1 to 3, wherein the cells are algae. A composition or kit for producing cells (excluding cells in a human individual) in which the methylation status of genomic DNA has been altered, the composition or kit comprising single-stranded DNA having a base sequence homologous to the base sequence of a DNA region in genomic DNA in which the methylation status is desired to be altered, and the kit does not contain DNA methylation enzyme. A composition or kit for producing cells (excluding cells in a human individual) in which the expression of a gene in genomic DNA is suppressed, the cells containing single-stranded DNA having a base sequence homologous to the base sequence of the sense strand of the gene whose expression is to be suppressed in the genomic DNA, and the cells not containing DNA methyltransferase.