WO2020111028A1 - Genome editing method - Google Patents

Genome editing method Download PDF

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Publication number
WO2020111028A1
WO2020111028A1 PCT/JP2019/046068 JP2019046068W WO2020111028A1 WO 2020111028 A1 WO2020111028 A1 WO 2020111028A1 JP 2019046068 W JP2019046068 W JP 2019046068W WO 2020111028 A1 WO2020111028 A1 WO 2020111028A1
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Prior art keywords
protein
gold particles
buffer solution
less
salt
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PCT/JP2019/046068
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French (fr)
Japanese (ja)
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晴康 濱田
洋三 柳楽
隆二 三木
直明 田岡
亮三 今井
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株式会社カネカ
国立研究開発法人農業・食品産業技術総合研究機構
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Priority to JP2020557718A priority Critical patent/JP7416383B2/en
Publication of WO2020111028A1 publication Critical patent/WO2020111028A1/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/14—Peptides being immobilised on, or in, an inorganic carrier
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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)

Definitions

  • the present invention relates to a technique for efficiently binding a protein to gold particles. More specifically, it relates to a technique for efficiently binding a protein to a gold particle used in a gene gun.
  • Genome editing technology is expected to be promising for modifying the genes of organisms (Patent Document 1).
  • Patent Document 1 Genome editing technology is expected to be promising for modifying the genes of organisms.
  • Patent Document 1 Genome editing technology is expected to be promising for modifying the genes of organisms.
  • Patent Document 1 Genome editing technology is expected to be promising for modifying the genes of organisms.
  • One of the reasons may be that a sufficient amount of CRISPR-CAS protein cannot be introduced into cells by a gene gun. Therefore, there has been a demand for a method that can introduce the CRISPR-CAS protein more efficiently.
  • a method for coating gold particles with a protein which comprises using one of the following buffers: 1) The final concentration of salt concentration excluding MgCl 2 and DTT in the buffer is 0 or less. A buffer that is at most 3.5 mM. 2) Salt-free buffer.
  • binding of protein to gold particles can be promoted.
  • FIG. 1 is a graph showing the relationship between the amount (%) of protein bound to gold particles and the salt concentration in various buffers.
  • FIG. 2 is a graph showing the effect of the salt concentration in buffer when the final concentration of Tris-HCl buffer is fixed at 1.0 mM on the amount of protein bound to gold particles.
  • FIG. 3 is a graph showing the effect of Tris-HCl concentration on the binding of protein to gold particles when the final concentration of NaCl is 1.0 mM.
  • FIG. 4 is a graph showing the effect of the MgCl 2 concentration in the buffer on the amount of protein bound to the gold particles.
  • the inventors of the present invention have conducted diligent research to use a buffer solution in which the final concentration of the salt concentration excluding MgCl 2 and DTT is more than 0 and 3.5 mM or less, or a buffer solution containing no salt.
  • the present invention has been completed by discovering that the efficiency of protein binding to gold particles can be increased by binding the protein to the gold particles. That is, according to the present invention, the binding of the protein to the gold particles can be promoted by using a buffer solution containing a salt at a concentration lower than usual. Coating gold particles using this method increases the binding rate of proteins to the gold particles to near 80%. When the final salt concentration is higher than 3.5 mM, the amount of protein bound to the gold particles is 55% or less (FIGS. 1 and 2).
  • the method for coating gold particles with a protein of the present invention is a method using any of the following buffers: 1) The final concentration of the salt concentration excluding MgCl 2 and DTT is more than 0 in the buffer. A buffer that is 3.5 mM or less. 2) Salt-free buffer.
  • the “salt” in the buffer solution means a salt that inhibits binding of protein to gold particles.
  • the “salt” includes, for example, sodium chloride (NaCl), potassium chloride (KCl), potassium acetate (KOAc), tris (Tris ⁇ Cl), Tris-OAc, HEPES, etc. Any substance that inhibits binding to gold particles is included without limitation. Note that magnesium chloride (MgCl 2 ) and dithiothreitol (DTT) are not included in the salt in this specification.
  • the salt preferably contains an alkali metal.
  • salt concentration refers to the concentration of salt that inhibits protein binding to gold particles.
  • the final salt concentration in the buffer solution is not particularly limited and may be appropriately selected depending on the intended purpose.
  • 2.5 mM or less is preferable, and 2.0 mM or less is more preferable.
  • Tris it is preferably 2.0 mM or less, and more preferably 1.5 mM or less, 1.0 mM or less, 0.5 mM or less, 0.3 mM or less.
  • the final concentration of the total salt concentration excluding MgCl 2 and DTT is preferably 3.5 mM or less, more preferably 3.0 mM or less, 2.5 mM or less, 2.0 mM or less, 1.5 mM or less, 1 0.0 mM or less, 0.5 mM or less, 0.3 mM or less, and may be 0 mM.
  • the buffer solution may further contain an amphipathic molecule and a basic protein.
  • the amphipathic molecule (compound) has a hydrophilic (water-soluble) portion and a hydrophobic (insoluble) portion.
  • Hydrophilic groups have the property that their chemical residues favor water. Hydrophilic groups include, but are not limited to, carbohydrates, polyoxyethylenes, peptides, oligonucleotides and groups containing amines, amides, alkoxyamides, carboxylic acids, sulfur, or hydroxyl groups.
  • Amphiphilic molecules (compounds) include, but are not limited to, lipid molecules, liposomes, lipopolyplexes, and the like.
  • the lipopolyplex may include, for example, 1,4-bis(3-oleoylamidopropyl)piperazine.
  • the hydrophobic group has the property that its chemical residue excludes water. Such chemical groups are not water soluble and do not form hydrogen bonds. Hydrocarbons are hydrophobic groups.
  • the amount of protein bound to the gold particle can be increased by using an amphipathic compound, and further, the release rate from the macrocarrier can be improved. ..
  • the amphipathic compound preferably has a cationic property.
  • the cationic amphiphilic compound may be a non-naturally occurring polyamine, the one or more amines being bound to at least one hydrophobic residue, which hydrophobic residue is C6. It may have a -C24 alkane, a C6-C24 alkene, a sterol, a steroid, a lipid, a fatty acid or a hydrophobic hormone.
  • the number X in CX represents the number of carbon atoms.
  • the amphipathic compound may or may not form liposomes.
  • the amphipathic compound may have the following structure.
  • R1 and R2 are substituents selected from the group consisting of C6-C24 alkanes, C6-C24 alkenes, sterols, steroids, lipids, fatty acids or hydrophobic hormones and other similar hydrophobic groups.
  • R1 and R2 may be the same or different.
  • the amphipathic compound may be 1,4-bis(3-oleoylamidopropyl)piperazine or Bis Imidazole ODAP (Bis imODAP, ODAP is Oxalyldiaminopropionic acid).
  • the concentration of the amphipathic molecule (compound) in the buffer solution is not particularly limited and may be appropriately selected depending on the intended purpose, but the final concentration is preferably 10 ⁇ g/mL or more, and 100 ⁇ g/mL or more. More preferable.
  • a polycation can be added in addition to the amphipathic compound.
  • Preferred polycations include, but are not limited to, polymers such as poly-L-lysine, polyethyleneimine (PEI), polysilazane, polydihydroimidazolenium, polyallylamine and similar compounds.
  • a preferred polycation is ethoxylated polyethyleneimine (ePEI).
  • the polycation may be a basic protein.
  • Suitable basic proteins are DNA binding proteins, eg histones such as H1, H2A or H2B. Histones may be derived from natural sources such as beef thymus, or may be recombinant proteins synthesized in bacteria. DNA binding proteins such as histones have various advantages over polycationic compounds such as polylysine.
  • the DNA binding protein may be a recombinant histone having both the SV40 large T antigen nuclear localization signal and the C-terminal domain of human histone H1 (NLS-H1), which is linked to the nuclear localization signal. There is.
  • the basic protein is not particularly limited as long as it improves the binding efficiency of the protein to the gold particles by mixing with 1,4-bis(3-oleoylamidopropyl)piperazine, but the histone H1 protein is preferable.
  • amphipathic compound and a polycation may be used in combination.
  • the ratio of 1,4-bis(3-oleoylamidopropyl)piperazine to histone H1 protein is preferably about 3:1.
  • the buffer solution may further include a nucleic acid.
  • the nucleic acid is appropriately selected depending on the intended purpose without any limitation, and examples thereof include DNA, RNA, and derivatives thereof.
  • the buffer solution may further contain other components.
  • the other components are appropriately selected depending on the intended purpose without any limitation, and examples thereof include MgCl 2 , DTT, bovine serum albumin (BSA), EDTA, DNase/RNase inhibitor, protease inhibitor, and glycerol. And so on.
  • the gold particles are not particularly limited and may be appropriately selected depending on the intended purpose. At least the surface may be gold, may have a solid structure, or may have a core-shell structure, or may be hollow. It may be particles or nanoparticles such as gold nanorods. The gold purity of the gold particles is appropriately selected depending on the intended purpose without any limitation, but it is preferably 99% or more.
  • the lower limit of the average particle size of the gold particles is not particularly limited and may be appropriately selected depending on the purpose, but is preferably 0.3 ⁇ m or more, more preferably 0.4 ⁇ m or more, and 0.5 ⁇ m or more. More preferably, 0.6 ⁇ m or more is particularly preferable.
  • the upper limit of the average particle size of the fine particles is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 1.5 ⁇ m or less, more preferably 1.4 ⁇ m or less, and even more preferably 1.3 ⁇ m or less. It is preferably 1.2 ⁇ m or less, particularly preferably 1.1 ⁇ m or less, and most preferably 1.0 ⁇ m or less.
  • the protein is appropriately selected depending on the intended purpose without any limitation, and examples thereof include nucleic acid metabolizing enzymes such as nuclease and deaminase.
  • the nuclease is appropriately selected depending on the intended purpose without any limitation, and examples thereof include CAS nuclease of CRISPR-CAS system.
  • the CAS nuclease is not particularly limited and may be appropriately selected depending on the intended purpose, and examples thereof include Cas I which is a type I CRISPR system enzyme and Cas9 which is a type II CRISPR system enzyme.
  • the concentration of the protein in the buffer solution is appropriately selected depending on the intended purpose without any limitation, but the final concentration is preferably 50 ⁇ g/mL or more, more preferably 300 ⁇ g/mL or more.
  • the lower limit of the coating time is appropriately selected depending on the intended purpose without any limitation, but it is preferably 3 minutes or longer, more preferably 5 minutes or longer, still more preferably 10 minutes or longer.
  • the upper limit of the coating time is appropriately selected depending on the intended purpose without any limitation, but it is preferably 30 minutes or less, more preferably 20 minutes or less, still more preferably 10 minutes or less.
  • the coating method is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a method in which a buffer solution and gold particles are mixed by pipetting or tapping, and the mixture is allowed to stand.
  • the method for measuring the amount of protein bound to the gold particles is not particularly limited and can be appropriately selected according to the purpose.
  • a mixed solution of gold particles and protein is centrifuged, and the supernatant is added to ⁇ 2.
  • SDS-PAGE sample buffer was added and heated at 95°C for 3 minutes.
  • CBB staining was performed and compared with the control (no gold particle) band, the protein considered to be adsorbed on the gold particle The amount can be calculated.
  • the final concentration of the salt concentration excluding MgCl 2 and DTT in the buffer solution can be measured by an ion chromatography method.
  • the ion chromatography method the relational expression between the concentration of salt excluding MgCl 2 and DTT and the response is obtained in advance, and the corresponding salt concentration is measured by applying the response obtained for the sample to the relational expression. can do.
  • the method for producing protein-coated gold particles of the present invention uses a buffer solution having a final salt concentration excluding MgCl 2 and DTT of more than 0 and 3.5 mM or less, or a salt-free buffer solution.
  • the method has a coating step of coating gold particles with a protein, and can further include other steps.
  • the coating step is the same as the method of coating the gold particles with a protein.
  • the method for producing a macrocarrier spotted with gold particles according to the present invention uses a buffer solution in which the final salt concentration excluding MgCl 2 and DTT is more than 0 and 3.5 mM or less, or a buffer solution containing no salt. Then, it has a coating step of coating the gold particles with a protein and a spotting step of spotting the gold particles obtained in the coating step on a macro carrier, and may further include other steps.
  • the coating step is the same as the method of coating the gold particles with a protein.
  • the gold particles are applied to the macro carrier film as evenly as possible using a pipette man or the like, and then dried in a sterile environment such as a clean bench.
  • the macro carrier film it is preferable to use a hydrophilic macro carrier film (“SH2CLHF” manufactured by 3M Co., Ltd.) or a macro carrier film coated with a hydrophilic coating agent.
  • Hydrophilic polymers used for hydrophilic coating of hydrophilic films and macrocarriers include polyethylene glycol, hydroxyethyl methacrylate, hydroxypropyl methacrylate, dihydroxyethyl methacrylate, diethylene glycol methacrylate, triethylene glycol methacrylate, polyethylene glycol methacrylate, vinylpyrrolidone, Polymers of hydrophilic monomers such as acrylic acid, acrylamide, dimethylacrylamide, glucooxyoxyethyl methacrylate, 3-sulfopropylmethacryloxyethyldimethylammonium betaine, 2-methacryloyloxyethylphosphorylcholine, 1-carboxydimethylmethacryloyloxyethylmethanammonium Can be mentioned.
  • a protein can be introduced into cells by a gene gun using the gold particles produced by the method for producing gold particles coated with protein or the macrocarrier produced by the method for producing macrocarriers spotted with the gold particles.
  • a protein can be introduced into cells by a gene gun using the gold particles produced by the method for producing gold particles coated with protein or the macrocarrier produced by the method for producing macrocarriers spotted with the gold particles.
  • the method of introducing the protein into the cells by the gene gun is not particularly limited and may be appropriately selected depending on the purpose.
  • a macrocarrier film, a plate on which a shoot apex of a target ripe embryo is placed is a particle gun.
  • a high-pressure helium gas is emitted from a gas accelerating tube toward a macrocarrier film by installing it in an apparatus.
  • Genome editing can be performed by the method of introducing a protein into a cell by the gene gun, and as a result, a genome-edited cell, tissue, organ, individual organism and/or progeny and/or seed can be obtained. ..
  • the organism for which the genome is to be edited is not particularly limited as long as it is an organism into which gold particles are introduced by a gene gun, and may be an animal, a plant, or a microorganism.
  • the plant may be a seed plant including angiosperms and gymnosperms, and the angiosperms include monocotyledons and dicotyledons.
  • the present invention can also be applied to the following plants.
  • the monocotyledon may be of any type, and examples thereof include Gramineae plants, Liliaceae plants, Musaceae plants, Pineapple plants and Orchidaceae plants.
  • the above Gramineae plants include rice, wheat, barley, corn, oats, turf, sorghum, rye, millet, sugar cane and the like.
  • Examples of the Liliaceae plant include leeks and asparagus.
  • Examples of the Musaceae plant include banana.
  • Examples of the pineapple plant include pineapple and the like.
  • Examples of the orchidaceous plant include orchids.
  • Examples of the dicotyledon for example, Brassicaceae, legumes, Solanaceae, Cucurbitaceae, Convolvulaceae, Rosaceae, Moraceae, Mallow, Asteraceae, Amaranthaceae, and Examples include Polygonaceae plants.
  • Examples of the cruciferous plant include Arabidopsis, Chinese cabbage, rapeseed, cabbage, cauliflower, and Japanese radish.
  • Examples of the legumes include soybean, adzuki bean, kidney bean, pea, cowpea, alfalfa and the like.
  • Examples of the Solanaceae plant include tomato, eggplant, potato, tobacco, and red pepper.
  • Examples of the Cucurbitaceae plant include makuwari, cucumber, melon and watermelon.
  • Examples of the bindweed plant include morning glory, sweet potato (sweet potato), bindweed, and the like.
  • Examples of the Rosaceae plant include roses, strawberries, apples and the like.
  • Examples of the mulberry plant include mulberry, fig, and rubber tree.
  • Examples of the mallow family plants include cotton and kenaf.
  • Examples of the Asteraceae plant include lettuce and the like.
  • Examples of the Amaranthaceae plant include sugar beet (sugar radish).
  • Examples of the Polygonaceae plant include buckwheat.
  • gymnosperms examples include pine, cedar, ginkgo and cycad.
  • the method of promoting the binding of the protein to the gold particles of the present invention is performed by using a buffer solution in which the final concentration of salt concentration excluding MgCl 2 and DTT is more than 0 and 3.5 mM or less, or a buffer solution containing no salt. It is a method of promoting the binding of proteins to gold particles.
  • the method of promoting the binding of the protein to the gold particles using the buffer solution is the same as the method of coating the gold particles with the protein using the buffer solution described above.
  • Fig. 1 is a plot of the binding amount (%) of the protein to the gold particles when various buffers were used. As shown in FIG. 1, A had the largest amount of binding to the gold particles. Since A had a NaCl concentration of 0 (see Table 1), it was considered that the lower the salt concentration, the greater the amount of protein bound to the gold particles.
  • Fig. 2 shows the amount of protein bound to gold particles when the NaCl concentration was changed at a final concentration of 1.0 mM TrisHCl (pH 7.5). As shown in FIG. 2, it can be seen that when the Tris concentration is constant, the binding amount of protein to the gold particles decreases as the NaCl concentration increases.
  • FIG. 3 shows the effect of the Tris concentration on the amount of protein bound to the gold particles.
  • the Tris concentration increased as the Tris concentration increased. It can be seen that the amount of protein bound is reduced.
  • Figure 4 is a MgCl 2, but in which was studied the effect on binding of protein to the gold particles, MgCl 2 is was not considered particularly affect the binding amount of the gold particles of the protein.
  • Examples of aspects of the present invention include the following.
  • a method for coating gold particles with a protein which comprises using one of the following buffers: 1) The final concentration of salt concentration excluding MgCl 2 and DTT in the buffer is A buffer solution that is greater than 0 and less than or equal to 3.5 mM. 2) Salt-free buffer.
  • a method for producing protein-coated gold particles which comprises a coating step of coating gold particles with protein using one of the following buffers: 1) A buffer solution in which the final concentration of the salt concentration excluding MgCl 2 and DTT in the buffer solution is more than 0 and 3.5 mM or less. 2) Salt-free buffer.
  • ⁇ 3> A method for producing a macro carrier in which gold particles are spotted, comprising a coating step of coating gold particles with a protein using one of the following buffer solutions, and the gold particles obtained in the coating step.
  • the method is characterized by comprising a spotting step of spotting on a macrocarrier: 1) A buffer solution in which the final salt concentration excluding MgCl 2 and DTT in the buffer solution is more than 0 and 3.5 mM or less. .. 2) Salt-free buffer.
  • ⁇ 4> A method of introducing a protein into a cell by a gene gun using the gold particles produced by the method described in ⁇ 2> or the macrocarrier produced by the method described in ⁇ 3>.
  • ⁇ 5> The method according to any one of ⁇ 1> to ⁇ 4>, wherein the buffer solution further contains an amphipathic molecule and a basic protein.
  • ⁇ 6> The method according to any one of ⁇ 1> to ⁇ 5>, wherein the buffer solution further contains a nucleic acid.
  • ⁇ 7> The method according to ⁇ 5> or ⁇ 6>, wherein the amphipathic molecule and the basic protein are 1,4-bis(3-oleoylamidopropyl)piperazine and histone H1.
  • ⁇ 8> A method for genome editing using the method according to any one of ⁇ 4> to ⁇ 7>.
  • ⁇ 9> A cell, tissue, organ, individual organism and/or progeny and/or seed obtained by the method according to any one of ⁇ 4> to ⁇ 8>.
  • ⁇ 10> A method of promoting the binding of a protein to gold particles using any of the following buffers: 1) The final concentration of salt concentration excluding MgCl 2 and DTT in the buffer is 0 A buffer that is more than 3.5 mM. 2) Salt-free buffer.
  • the present invention can be used in the biotechnology industry, agriculture, breed breeding industry, etc.

Abstract

A method in which gold particles are coated with protein, the method being characterized by using either of the following buffering solutions. 1) A buffering solution that has a final salt concentration, excluding MgCl2 and DTT, of greater than 0 mM but no greater than 3.5 mM. 2) A buffering solution that does not include a salt.

Description

ゲノム編集方法Genome editing method
　本発明は、タンパク質を金粒子に効率よく結合させる技術に関する。より詳しくは、遺伝子銃に使用する金粒子にタンパク質を効率よく結合させる技術に関する。 The present invention relates to a technique for efficiently binding a protein to gold particles. More specifically, it relates to a technique for efficiently binding a protein to a gold particle used in a gene gun.
　生物の遺伝子を改変するために、ゲノム編集技術が有望であると期待されている（特許文献１）。
　しかしながら、現実には、ゲノム編集が難しい植物も存在している。その理由の１つとしては、充分な量のＣＲＩＳＰＲ－ＣＡＳタンパク質を遺伝子銃により細胞中に導入できないことが考えられる。
　そこで、より効率よくＣＲＩＳＰＲ－ＣＡＳタンパク質を導入できる方法が求められていた。 Genome editing technology is expected to be promising for modifying the genes of organisms (Patent Document 1).
However, in reality, there are plants in which genome editing is difficult. One of the reasons may be that a sufficient amount of CRISPR-CAS protein cannot be introduced into cells by a gene gun.
Therefore, there has been a demand for a method that can introduce the CRISPR-CAS protein more efficiently.
米国特許公開公報第２０１４／００６８７９７号US Patent Publication No. 2014/0068797
　タンパク質を金粒子に効率よく被覆する方法を提供する。 Provide a method for efficiently coating gold particles with protein.
　前記課題を解決するための手段としては、以下の通りである。即ち、
　＜１＞　以下のいずれかの緩衝液を用いることを特徴とする、金粒子をタンパク質によりコーティングする方法：１）前記緩衝液における、ＭｇＣｌ２、及びＤＴＴを除く塩濃度の終濃度が、０より多く３．５ｍＭ以下である緩衝液。２）塩を含まない緩衝液。 The means for solving the above problems are as follows. That is,
<1> A method for coating gold particles with a protein, which comprises using one of the following buffers: 1) The final concentration of salt concentration excluding MgCl 2 and DTT in the buffer is 0 or less. A buffer that is at most 3.5 mM. 2) Salt-free buffer.
　本発明によれば、タンパク質の金粒子への結合を促進することができる。 According to the present invention, binding of protein to gold particles can be promoted.
図１は、金粒子へのタンパク質の結合量（％）と種々のＢｕｆｆｅｒ中の塩濃度の関係を示すグラフである。FIG. 1 is a graph showing the relationship between the amount (%) of protein bound to gold particles and the salt concentration in various buffers. 図２は、Ｔｒｉｓ－ＨＣｌ　ｂｕｆｆｅｒの終濃度を１．０ｍＭに固定した場合のｂｕｆｆｅｒ中の塩濃度が、タンパク質の金粒子への結合量に及ぼす影響を示すグラフである。FIG. 2 is a graph showing the effect of the salt concentration in buffer when the final concentration of Tris-HCl buffer is fixed at 1.0 mM on the amount of protein bound to gold particles. 図３は、ＮａＣｌの終濃度が１．０ｍＭの場合に、Ｔｒｉｓ－ＨＣｌ濃度が、タンパク質の金粒子への結合に及ぼす影響を示すグラフである。FIG. 3 is a graph showing the effect of Tris-HCl concentration on the binding of protein to gold particles when the final concentration of NaCl is 1.0 mM. 図４は、ｂｕｆｆｅｒ中のＭｇＣｌ２濃度が、タンパク質の金粒子への結合量に及ぼす影響を示すグラフである。FIG. 4 is a graph showing the effect of the MgCl 2 concentration in the buffer on the amount of protein bound to the gold particles.
　ＣＲＩＳＰＲ－ＣＡＳを用いてゲノム編集をする場合に遺伝子銃を用いてＲＮＡとタンパク質を導入する方法がある。しかしながら、遺伝子銃を用いてタンパク質を導入する場合、様々な問題がある。１つは、ＤＮＡと異なり、変性しやすく親水性であるタンパク質を金粒子に結合させる効率の問題、もう１つは、タンパク質を結合させた金粒子をマクロキャリアにスポットする際の問題、マクロキャリア上にスポットした金粒子がマクロキャリアからどの程度の割合で放出されるかの問題などである。 There is a method of introducing RNA and protein using a gene gun when editing the genome using CRISPR-CAS. However, there are various problems when introducing proteins using a gene gun. One is the problem of the efficiency of binding the protein, which is different from DNA and is easily denatured and hydrophilic, to the gold particles, and the other is the problem of spotting the protein-bound gold particles on the macrocarrier, the macrocarrier. There is a problem such as how much the gold particles spotted on the top are released from the macrocarrier.
　そこで、本発明者らは、鋭意研究することにより、ＭｇＣｌ２、及びＤＴＴを除く塩濃度の終濃度が、０より多く３．５ｍＭ以下である緩衝液、又は塩を含まない緩衝液を用いて、金粒子にタンパク質を結合させることにより、タンパク質の金粒子への結合効率を高められることを発見し、本発明を完成した。すなわち、本発明によれば、通常よりも低濃度の塩を含む緩衝液を用いることで、タンパク質の金粒子への結合を促進することができる。この方法を用いて金粒子をコーティングすることにより、金粒子へのタンパク質の結合率が約８０％近くにまで上昇する。塩濃度の終濃度が３．５ｍＭより高い場合、金粒子へのタンパク質の結合量は５５％以下である（図１および図２）。 Therefore, the inventors of the present invention have conducted diligent research to use a buffer solution in which the final concentration of the salt concentration excluding MgCl 2 and DTT is more than 0 and 3.5 mM or less, or a buffer solution containing no salt. The present invention has been completed by discovering that the efficiency of protein binding to gold particles can be increased by binding the protein to the gold particles. That is, according to the present invention, the binding of the protein to the gold particles can be promoted by using a buffer solution containing a salt at a concentration lower than usual. Coating gold particles using this method increases the binding rate of proteins to the gold particles to near 80%. When the final salt concentration is higher than 3.5 mM, the amount of protein bound to the gold particles is 55% or less (FIGS. 1 and 2).
（金粒子をタンパク質によりコーティングする方法）
　本発明の金粒子をタンパク質によりコーティングする方法は、以下のいずれかの緩衝液を用いる方法である：１）前記緩衝液における、ＭｇＣｌ２、及びＤＴＴを除く塩濃度の終濃度が、０より多く３．５ｍＭ以下である緩衝液。２）塩を含まない緩衝液。 (Method of coating gold particles with protein)
The method for coating gold particles with a protein of the present invention is a method using any of the following buffers: 1) The final concentration of the salt concentration excluding MgCl 2 and DTT is more than 0 in the buffer. A buffer that is 3.5 mM or less. 2) Salt-free buffer.
　前記緩衝液における「塩」とは、タンパク質が金粒子に結合するのを阻害する塩をいう。「塩」とは、例えば、食塩（ＮａＣｌ）、塩化カリウム（ＫＣｌ）、酢酸カリウム（ＫＯＡｃ）、トリス（Ｔｒｉｓ・Ｃｌ）、Ｔｒｉｓ－ＯＡｃ、ＨＥＰＥＳ等が含まれるが、これらに限られず、タンパク質が金粒子に結合するのを阻害する物質であれば制限なく含まれる。なお、塩化マグネシウム（ＭｇＣｌ２）、及びジチオトレイトール（ＤＴＴ）は本明細書においては、塩に含まれない。塩としては、好ましくはアルカリ金属を含む。本明細書において、「塩濃度」とは、タンパク質が金粒子に結合するのを阻害する塩の濃度をいう。 The “salt” in the buffer solution means a salt that inhibits binding of protein to gold particles. The “salt” includes, for example, sodium chloride (NaCl), potassium chloride (KCl), potassium acetate (KOAc), tris (Tris·Cl), Tris-OAc, HEPES, etc. Any substance that inhibits binding to gold particles is included without limitation. Note that magnesium chloride (MgCl 2 ) and dithiothreitol (DTT) are not included in the salt in this specification. The salt preferably contains an alkali metal. As used herein, the term "salt concentration" refers to the concentration of salt that inhibits protein binding to gold particles.
　前記緩衝液における好ましい塩濃度の終濃度としては、特に制限はなく、目的に応じて適宜選択することができるが、食塩の場合、２．５ｍＭ以下が好ましく、より好ましくは２．０ｍＭ以下、１．５ｍＭ以下、１．０ｍＭ以下、０．５ｍＭ以下、０．３ｍＭ以下、０．１ｍＭ以下であり、最も好ましくは０ｍＭである。Ｔｒｉｓの場合は、２．０ｍＭ以下が好ましく、より好ましくは、１．５ｍＭ以下、１．０ｍＭ以下、０．５ｍＭ以下、０．３ｍＭ以下であってもよい。また、ＭｇＣｌ２、及びＤＴＴを除く全体の塩濃度の終濃度としては、３．５ｍＭ以下が好ましく、より好ましくは３．０ｍＭ以下、２．５ｍＭ以下、２．０ｍＭ以下、１．５ｍＭ以下、１．０ｍＭ以下、０．５ｍＭ以下、０．３ｍＭ以下であり、０ｍＭであっても良い。 The final salt concentration in the buffer solution is not particularly limited and may be appropriately selected depending on the intended purpose. In the case of salt, 2.5 mM or less is preferable, and 2.0 mM or less is more preferable. 0.5 mM or less, 1.0 mM or less, 0.5 mM or less, 0.3 mM or less, 0.1 mM or less, and most preferably 0 mM. In the case of Tris, it is preferably 2.0 mM or less, and more preferably 1.5 mM or less, 1.0 mM or less, 0.5 mM or less, 0.3 mM or less. Further, the final concentration of the total salt concentration excluding MgCl 2 and DTT is preferably 3.5 mM or less, more preferably 3.0 mM or less, 2.5 mM or less, 2.0 mM or less, 1.5 mM or less, 1 0.0 mM or less, 0.5 mM or less, 0.3 mM or less, and may be 0 mM.
　前記緩衝液は、さらに、両親媒性分子及び塩基性タンパク質を含むことができる。両親媒性分子（化合物）は、親水性（水溶性）部分と、疎水性（不溶性）部分とを有している。親水性基は、その化学残基が水を好む性質を有している。親水性基としては、例えば、炭水化物、ポリオキシエチレン、ペプチド、オリゴヌクレオチド並びにアミン、アミド、アルコキシアミド、カルボン酸、イオウ、又は水酸基を含む基が含まれるが、これらに限られない。両親媒性分子（化合物）としては、脂質分子、リポソーム、リポポリプレックス（ｌｉｐｏ　ｐｏｌｙｐｌｅｘ）などを含むがこれらに限られない。リポポリプレックスは、例えば、１，４－ビス（３－オレオイルアミドプロピル）ピペラジンを含んでいてもよい。 The buffer solution may further contain an amphipathic molecule and a basic protein. The amphipathic molecule (compound) has a hydrophilic (water-soluble) portion and a hydrophobic (insoluble) portion. Hydrophilic groups have the property that their chemical residues favor water. Hydrophilic groups include, but are not limited to, carbohydrates, polyoxyethylenes, peptides, oligonucleotides and groups containing amines, amides, alkoxyamides, carboxylic acids, sulfur, or hydroxyl groups. Amphiphilic molecules (compounds) include, but are not limited to, lipid molecules, liposomes, lipopolyplexes, and the like. The lipopolyplex may include, for example, 1,4-bis(3-oleoylamidopropyl)piperazine.
　疎水性基は、その化学残基は水を排除する性質を有している。かかる化学基は水溶性ではなく、水素結合を形成しない。炭化水素は、疎水性基である。 　The hydrophobic group has the property that its chemical residue excludes water. Such chemical groups are not water soluble and do not form hydrogen bonds. Hydrocarbons are hydrophobic groups.
　本発明の金粒子のタンパク質被覆方法においては、両親媒性化合物を用いることにより、金粒子へのタンパク質の結合量を増加させることができ、さらに、マクロキャリアからの放出率も向上させることができる。 In the method for coating a gold particle with a protein of the present invention, the amount of protein bound to the gold particle can be increased by using an amphipathic compound, and further, the release rate from the macrocarrier can be improved. ..
　両親媒性化合物は陽イオン性を有していることが好ましい。この陽イオン性両親媒性化合物は、天然由来ではないポリアミンであってもよく、これら１つ以上のアミンは、少なくとも１つの疎水性残基に結合されており、この疎水性残基は、Ｃ６－Ｃ２４アルカン、Ｃ６－Ｃ２４アルケン、ステロール、ステロイド、脂質、脂肪酸又は疎水性ホルモンを有していてもよい。ここでＣＸのＸの数字は炭素数を表す。両親媒性化合物は、リポソームを形成していても形成していなくてもよい。また、両親媒性化合物は、以下の構造を有していてもよい。 The amphipathic compound preferably has a cationic property. The cationic amphiphilic compound may be a non-naturally occurring polyamine, the one or more amines being bound to at least one hydrophobic residue, which hydrophobic residue is C6. It may have a -C24 alkane, a C6-C24 alkene, a sterol, a steroid, a lipid, a fatty acid or a hydrophobic hormone. Here, the number X in CX represents the number of carbon atoms. The amphipathic compound may or may not form liposomes. In addition, the amphipathic compound may have the following structure.
Figure JPOXMLDOC01-appb-C000001
Figure JPOXMLDOC01-appb-C000001
　Ｒ１及びＲ２は、Ｃ６－Ｃ２４アルカン、Ｃ６－Ｃ２４アルケン、ステロール、ステロイド、脂質、脂肪酸又は疎水性ホルモン及びその他の類似する疎水基からなる群から選択された置換基である。Ｒ１及びＲ２は、同一であっても異なっていてもよい。
　両親媒性化合物は、１，４－ビス（３－オレオイルアミドプロピル）ピペラジンまたはＢｉｓ　Ｉｍｉｄａｚｏｌｅ　ＯＤＡＰ　（Ｂｉｓ　ｉｍＯＤＡＰ，　ＯＤＡＰはＯｘａｌｙｌｄｉａｍｉｎｏｐｒｏｐｉｏｎｉｃ　ａｃｉｄ）であってもよい。 R1 and R2 are substituents selected from the group consisting of C6-C24 alkanes, C6-C24 alkenes, sterols, steroids, lipids, fatty acids or hydrophobic hormones and other similar hydrophobic groups. R1 and R2 may be the same or different.
The amphipathic compound may be 1,4-bis(3-oleoylamidopropyl)piperazine or Bis Imidazole ODAP (Bis imODAP, ODAP is Oxalyldiaminopropionic acid).
　前記両親媒性分子（化合物）の、前記緩衝液における濃度としては、特に制限はなく、目的に応じて適宜選択することができるが、終濃度が１０μｇ／ｍＬ以上が好ましく、１００μｇ／ｍＬ以上がより好ましい。 The concentration of the amphipathic molecule (compound) in the buffer solution is not particularly limited and may be appropriately selected depending on the intended purpose, but the final concentration is preferably 10 μg/mL or more, and 100 μg/mL or more. More preferable.
　本発明においては、両親媒性化合物に加えて、ポリカチオンを添加することができる。好ましいポリカチオンとしては、ポリ－Ｌ－リジン、ポリエチレンイミン（ＰＥＩ）、ポリシラザン、ポリジヒドロイミダゾレニウム、ポリアリルアミン及びこれらに類する化合物などのポリマーなどが挙げられるがこれらに限られない。好適なポリカチオンは、エトキシル化ポリエチレンイミン（ｅＰＥＩ）である。 In the present invention, a polycation can be added in addition to the amphipathic compound. Preferred polycations include, but are not limited to, polymers such as poly-L-lysine, polyethyleneimine (PEI), polysilazane, polydihydroimidazolenium, polyallylamine and similar compounds. A preferred polycation is ethoxylated polyethyleneimine (ePEI).
　また、ポリカチオンは塩基性タンパク質であってもよい。好適な塩基性タンパク質は、ＤＮＡ結合タンパク質であり、例えばＨ１、Ｈ２Ａ又はＨ２Ｂなどのヒストンである。ヒストンは、牛胸腺などの天然ソースに由来していてもよいし、バクテリア内で合成された組換えタンパク質であってもよい。ヒストンなどのＤＮＡ結合タンパクは、ポリリジンなどのポリカチオン化合物に比べて種々の点で利点を有している。ＤＮＡ結合タンパク質は、ＳＶ４０ラージＴ抗原核局在化シグナルやヒトヒストンＨ１のＣ末端ドメイン（ＮＬＳ－Ｈ１）の両方を有する組換えヒストンであってもよく、これは核局在化シグナルにリンクされている。塩基性タンパク質は、１，４－ビス（３－オレオイルアミドプロピル）ピペラジンと混合することでタンパク質の金粒子への結合効率が上がるものであれば特に限定されないが、ヒストンＨ１タンパク質が好ましい。 Also, the polycation may be a basic protein. Suitable basic proteins are DNA binding proteins, eg histones such as H1, H2A or H2B. Histones may be derived from natural sources such as beef thymus, or may be recombinant proteins synthesized in bacteria. DNA binding proteins such as histones have various advantages over polycationic compounds such as polylysine. The DNA binding protein may be a recombinant histone having both the SV40 large T antigen nuclear localization signal and the C-terminal domain of human histone H1 (NLS-H1), which is linked to the nuclear localization signal. There is. The basic protein is not particularly limited as long as it improves the binding efficiency of the protein to the gold particles by mixing with 1,4-bis(3-oleoylamidopropyl)piperazine, but the histone H1 protein is preferable.
　また、両親媒性化合物と、ポリカチオンを組み合わせて用いてもよい。例えば、１，４－ビス（３－オレオイルアミドプロピル）ピペラジンとヒストンＨ１タンパク質の比率は、３：１程度が好ましい。 Alternatively, an amphipathic compound and a polycation may be used in combination. For example, the ratio of 1,4-bis(3-oleoylamidopropyl)piperazine to histone H1 protein is preferably about 3:1.
　前記緩衝液は、さらに、核酸を含むことができる。
　前記核酸としては、特に制限はなく、目的に応じて適宜選択することができ、ＤＮＡ、ＲＮＡ、及びこれらの誘導体などが挙げられる。 The buffer solution may further include a nucleic acid.
The nucleic acid is appropriately selected depending on the intended purpose without any limitation, and examples thereof include DNA, RNA, and derivatives thereof.
　前記緩衝液は、さらに、その他の成分を含むことができる。
　前記その他の成分としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、ＭｇＣｌ２、ＤＴＴ、ウシ血清アルブミン（ＢＳＡ）、ＥＤＴＡ、ＤＮａｓｅ/ＲＮａｓｅ阻害剤、プロテアーゼ阻害剤、グリセロールなどが挙げられる。 The buffer solution may further contain other components.
The other components are appropriately selected depending on the intended purpose without any limitation, and examples thereof include MgCl 2 , DTT, bovine serum albumin (BSA), EDTA, DNase/RNase inhibitor, protease inhibitor, and glycerol. And so on.
　前記金粒子としては、特に制限はなく、目的に応じて適宜選択することができ、少なくとも表面が金であればよく、中実構造のものでもよいし、コアシェル構造のものであっても、中空粒子であってもよく、金ナノロッドなどのナノ粒子であってもよい。
　前記金粒子の金の純度としては、特に制限はなく、目的に応じて適宜選択することができるが、９９％以上が好ましい。 The gold particles are not particularly limited and may be appropriately selected depending on the intended purpose. At least the surface may be gold, may have a solid structure, or may have a core-shell structure, or may be hollow. It may be particles or nanoparticles such as gold nanorods.
The gold purity of the gold particles is appropriately selected depending on the intended purpose without any limitation, but it is preferably 99% or more.
　前記金粒子の平均粒径の下限値としては、特に制限はなく、目的に応じて適宜選択することができるが、０．３μｍ以上が好ましく、０．４μｍ以上がより好ましく、０．５μｍ以上がさらに好ましく、０．６μｍ以上が特に好ましい。
　前記微粒子の平均粒径の上限値としては、特に制限はなく、目的に応じて適宜選択することができるが、１．５μｍ以下が好ましく、１．４μｍ以下がより好ましく、１．３μｍ以下がさらに好ましく、１．２μｍ以下が特に好ましく、１．１μｍ以下がさらに特に好ましく、１．０μｍ以下が最も好ましい。 The lower limit of the average particle size of the gold particles is not particularly limited and may be appropriately selected depending on the purpose, but is preferably 0.3 μm or more, more preferably 0.4 μm or more, and 0.5 μm or more. More preferably, 0.6 μm or more is particularly preferable.
The upper limit of the average particle size of the fine particles is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 1.5 μm or less, more preferably 1.4 μm or less, and even more preferably 1.3 μm or less. It is preferably 1.2 μm or less, particularly preferably 1.1 μm or less, and most preferably 1.0 μm or less.
　前記タンパクとしては、特に制限はなく、目的に応じて適宜選択することができ、例えば、ヌクレアーゼ、又はデアミナーゼなどの核酸代謝酵素が挙げられる。
　前記ヌクレアーゼとしては、特に制限はなく、目的に応じて適宜選択することができ、例えば、ＣＲＩＳＰＲ－ＣＡＳシステムのＣＡＳヌクレアーゼなどが挙げられる。
　前記ＣＡＳヌクレアーゼとしては、特に制限はなく、目的に応じて適宜選択することができ、例えば、Ｉ型ＣＲＩＳＰＲ系酵素であるＣａｓ３、ＩＩ型ＣＲＩＳＰＲ系酵素であるＣａｓ９などが挙げられる。 The protein is appropriately selected depending on the intended purpose without any limitation, and examples thereof include nucleic acid metabolizing enzymes such as nuclease and deaminase.
The nuclease is appropriately selected depending on the intended purpose without any limitation, and examples thereof include CAS nuclease of CRISPR-CAS system.
The CAS nuclease is not particularly limited and may be appropriately selected depending on the intended purpose, and examples thereof include Cas I which is a type I CRISPR system enzyme and Cas9 which is a type II CRISPR system enzyme.
　前記タンパクの、前記緩衝液における濃度としては、特に制限はなく、目的に応じて適宜選択することができるが、終濃度が５０μｇ／ｍＬ以上が好ましく、３００μｇ／ｍＬ以上がより好ましい。 The concentration of the protein in the buffer solution is appropriately selected depending on the intended purpose without any limitation, but the final concentration is preferably 50 µg/mL or more, more preferably 300 µg/mL or more.
　前記コーティングの時間の下限値としては、特に制限はなく、目的に応じて適宜選択することができるが、３分間以上が好ましく、５分間以上がより好ましく、１０分間以上がさらに好ましい。
　前記コーティングの時間の上限値としては、特に制限はなく、目的に応じて適宜選択することができるが、３０分間以下が好ましく、２０分間以下がより好ましく、１０分間以下がさらに好ましい。 The lower limit of the coating time is appropriately selected depending on the intended purpose without any limitation, but it is preferably 3 minutes or longer, more preferably 5 minutes or longer, still more preferably 10 minutes or longer.
The upper limit of the coating time is appropriately selected depending on the intended purpose without any limitation, but it is preferably 30 minutes or less, more preferably 20 minutes or less, still more preferably 10 minutes or less.
　前記コーティングの方法としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、緩衝液と金粒子をピペッティングまたはタッピングにより混合し、静置する方法などが挙げられる。 The coating method is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a method in which a buffer solution and gold particles are mixed by pipetting or tapping, and the mixture is allowed to stand.
　前記金粒子へのタンパク質の結合量の測定方法としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、金粒子とタンパクの混合液を遠心し、上清に、×２　ＳＤＳ－ＰＡＧＥ用ｓａｍｐｌｅ　ｂｕｆｆｅｒを加え、９５℃３分間加熱し、ＳＤＳ－ＰＡＧＥによりタンパク質を分離後、ＣＢＢ染色し、コントロール(金粒子なし)のバンドと比較し、金粒子に吸着したと考えられるタンパク質量を算出することができる。 The method for measuring the amount of protein bound to the gold particles is not particularly limited and can be appropriately selected according to the purpose. For example, a mixed solution of gold particles and protein is centrifuged, and the supernatant is added to ×2. SDS-PAGE sample buffer was added and heated at 95°C for 3 minutes. After separating the protein by SDS-PAGE, CBB staining was performed and compared with the control (no gold particle) band, the protein considered to be adsorbed on the gold particle The amount can be calculated.
　前記緩衝液における、ＭｇＣｌ２、及びＤＴＴを除く塩濃度の終濃度は、イオンクロマトグラフ法により測定することができる。前記イオンクロマトグラフ法では、あらかじめＭｇＣｌ２、及びＤＴＴを除く塩の濃度と応答との関係式を求めておき、サンプルについて得られた応答をその関係式に当てはめることで、該当する塩濃度を測定することができる。 The final concentration of the salt concentration excluding MgCl 2 and DTT in the buffer solution can be measured by an ion chromatography method. In the ion chromatography method, the relational expression between the concentration of salt excluding MgCl 2 and DTT and the response is obtained in advance, and the corresponding salt concentration is measured by applying the response obtained for the sample to the relational expression. can do.
（タンパク質がコーティングされた金粒子の製造方法）
　本発明のタンパク質がコーティングされた金粒子の製造方法は、ＭｇＣｌ２、及びＤＴＴを除く塩濃度の終濃度が、０より多く３．５ｍＭ以下である緩衝液、又は塩を含まない緩衝液を用いて、金粒子をタンパク質によりコーティングするコーティング工程を有し、さらにその他の工程を含むことができる。
　前記コーティング工程は、前述の金粒子をタンパク質によりコーティングする方法のとおりである。 (Method for producing gold particles coated with protein)
The method for producing protein-coated gold particles of the present invention uses a buffer solution having a final salt concentration excluding MgCl 2 and DTT of more than 0 and 3.5 mM or less, or a salt-free buffer solution. Thus, the method has a coating step of coating gold particles with a protein, and can further include other steps.
The coating step is the same as the method of coating the gold particles with a protein.
（金粒子をスポットしたマクロキャリアの製造方法）
　本発明の金粒子をスポットしたマクロキャリアの製造方法は、ＭｇＣｌ２、及びＤＴＴを除く塩濃度の終濃度が、０より多く３．５ｍＭ以下である緩衝液、又は塩を含まない緩衝液を用いて、金粒子をタンパク質によりコーティングするコーティング工程、及び、前記コーティング工程で得られた金粒子をマクロキャリアにスポットするスポット工程を有し、さらにその他の工程を含むことができる。
　前記コーティング工程は、前述の金粒子をタンパク質によりコーティングする方法のとおりである。 (Manufacturing method of macro carrier with spotted gold particles)
The method for producing a macrocarrier spotted with gold particles according to the present invention uses a buffer solution in which the final salt concentration excluding MgCl 2 and DTT is more than 0 and 3.5 mM or less, or a buffer solution containing no salt. Then, it has a coating step of coating the gold particles with a protein and a spotting step of spotting the gold particles obtained in the coating step on a macro carrier, and may further include other steps.
The coating step is the same as the method of coating the gold particles with a protein.
　前記スポット工程において、金粒子は、ピペットマンなどを用いてマクロキャリヤーフィルムに可能な限り均一に塗布した後、クリーンベンチなどの無菌環境中で乾燥させる。前記マクロキャリヤーフィルムとしては、親水性のマクロキャリヤーフィルム（３Ｍ社「ＳＨ２ＣＬＨＦ」など）、又は、親水性のコーティング剤によりコーティングしたマクロキャリヤーフィルムを用いるのが好ましい。 In the spotting process, the gold particles are applied to the macro carrier film as evenly as possible using a pipette man or the like, and then dried in a sterile environment such as a clean bench. As the macro carrier film, it is preferable to use a hydrophilic macro carrier film (“SH2CLHF” manufactured by 3M Co., Ltd.) or a macro carrier film coated with a hydrophilic coating agent.
　親水性フィルムやマクロキャリアの親水性のコーティングに用いられる親水性ポリマーとしては、ポリエチレングリコール、ヒドロキシエチルメタクリレート、ヒドロキシプロピルメタクリレート、ジヒドロキシエチルメタクリレート、ジエチレングリコールメタクリレート、トリエチレングリコールメタクリレート、ポリエチレングリコールメタクリレート、ビニルピロリドン、アクリル酸、アクリルアミド、ジメチルアクリルアミド、グルコキシオキシエチルメタクリレート、３－スルホプロピルメタクリルオキシエチルジメチルアンモニウムベタイン、２－メタクリロイルオキシエチルホスホリルコリン、１－カルボキシジメチルメタクリロイルオキシエチルメタンアンモニウム等の親水性モノマーの重合体が挙げられる。 Hydrophilic polymers used for hydrophilic coating of hydrophilic films and macrocarriers include polyethylene glycol, hydroxyethyl methacrylate, hydroxypropyl methacrylate, dihydroxyethyl methacrylate, diethylene glycol methacrylate, triethylene glycol methacrylate, polyethylene glycol methacrylate, vinylpyrrolidone, Polymers of hydrophilic monomers such as acrylic acid, acrylamide, dimethylacrylamide, glucooxyoxyethyl methacrylate, 3-sulfopropylmethacryloxyethyldimethylammonium betaine, 2-methacryloyloxyethylphosphorylcholine, 1-carboxydimethylmethacryloyloxyethylmethanammonium Can be mentioned.
（遺伝子銃により細胞にタンパク質を導入する方法）
　前記タンパク質がコーティングされた金粒子の製造方法で製造した金粒子、または前記金粒子をスポットしたマクロキャリアの製造方法で製造したマクロキャリアを用いて、遺伝子銃により細胞にタンパク質を導入することができ、その結果、タンパク質が導入された細胞、組織、器官、生物個体ならびに／またはその後代および／もしくは種子を得ることができる。 (Method of introducing protein into cells by gene gun)
A protein can be introduced into cells by a gene gun using the gold particles produced by the method for producing gold particles coated with protein or the macrocarrier produced by the method for producing macrocarriers spotted with the gold particles. As a result, cells, tissues, organs, individual organisms and/or progeny and/or seeds into which the protein has been introduced can be obtained.
　前記遺伝子銃により細胞にタンパク質を導入する方法としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、マクロキャリヤーフィルム、ターゲットの完熟胚の茎頂を置床したプレートをパーティクルガン装置に設置し、ガス加速管から高圧ヘリウムガスをマクロキャリヤーフィルムに向かって発射する方法などが挙げられる。 The method of introducing the protein into the cells by the gene gun is not particularly limited and may be appropriately selected depending on the purpose.For example, a macrocarrier film, a plate on which a shoot apex of a target ripe embryo is placed is a particle gun. There is a method in which a high-pressure helium gas is emitted from a gas accelerating tube toward a macrocarrier film by installing it in an apparatus.
（ゲノム編集を行う方法）
　前記遺伝子銃により細胞にタンパク質を導入する方法により、ゲノム編集を行うことができ、その結果、ゲノム編集された細胞、組織、器官、生物個体ならびに／またはその後代および／もしくは種子を得ることができる。 (Method to edit genome)
Genome editing can be performed by the method of introducing a protein into a cell by the gene gun, and as a result, a genome-edited cell, tissue, organ, individual organism and/or progeny and/or seed can be obtained. ..
　前記ゲノム編集の対象となる生物は、遺伝子銃により金粒子が導入される生物であれば、特に制限されず、動物、植物、微生物であってもよい。前記植物としては、被子植物及び裸子植物を含む種子植物であってもよく、前記被子植物には、単子葉植物及び双子葉植物が含まれる。また、以下の植物についても本発明を適用し得る。 The organism for which the genome is to be edited is not particularly limited as long as it is an organism into which gold particles are introduced by a gene gun, and may be an animal, a plant, or a microorganism. The plant may be a seed plant including angiosperms and gymnosperms, and the angiosperms include monocotyledons and dicotyledons. The present invention can also be applied to the following plants.
　前記単子葉植物としては、いずれの種類であってもよいが、例えば、イネ科植物、ユリ科植物、バショウ科植物、パイナップル科植物、ラン科植物などが挙げられる。 The monocotyledon may be of any type, and examples thereof include Gramineae plants, Liliaceae plants, Musaceae plants, Pineapple plants and Orchidaceae plants.
　前記イネ科植物としては、イネ、コムギ、オオムギ、トウモロコシ、エンバク、シバ、ソルガム、ライムギ、アワ、サトウキビなどが挙げられる。前記ユリ科植物としては、ネギ、アスパラガスなどが挙げられる。前記バショウ科植物としては、バナナなどが挙げられる。前記パイナップル科植物としては、パイナップルなどが挙げられる。前記ラン科植物としては、ランなどが挙げられる。 The above Gramineae plants include rice, wheat, barley, corn, oats, turf, sorghum, rye, millet, sugar cane and the like. Examples of the Liliaceae plant include leeks and asparagus. Examples of the Musaceae plant include banana. Examples of the pineapple plant include pineapple and the like. Examples of the orchidaceous plant include orchids.
　前記双子葉植物としては、例えば、アブラナ科植物、マメ科植物、ナス科植物、ウリ科植物、ヒルガオ科植物、バラ科植物、クワ科植物、アオイ科植物、キク科植物、ヒユ科植物、およびタデ科植物などが挙げられる。 Examples of the dicotyledon, for example, Brassicaceae, legumes, Solanaceae, Cucurbitaceae, Convolvulaceae, Rosaceae, Moraceae, Mallow, Asteraceae, Amaranthaceae, and Examples include Polygonaceae plants.
　前記アブラナ科植物としては、シロイヌナズナ、ハクサイ、ナタネ、キャベツ、カリフラワー、ダイコンなどが挙げられる。前記マメ科植物としては、ダイズ、アズキ、インゲンマメ、エンドウ、ササゲ、アルファルファなどが挙げられる。前記ナス科植物としては、トマト、ナス、ジャガイモ、タバコ、トウガラシなどが挙げられる。前記ウリ科植物としては、マクワウリ、キュウリ、メロン、スイカなどが挙げられる。前記ヒルガオ科植物としては、アサガオ、サツマイモ（カンショ）、ヒルガオなどが挙げられる。前記バラ科植物としては、バラ、イチゴ、リンゴなどが挙げられる。前記クワ科植物としては、クワ、イチジク、ゴムノキなどが挙げられる。前記アオイ科植物としては、ワタ、ケナフなどが挙げられる。前記キク科植物としては、レタスなどが挙げられる。前記ヒユ科植物としては、テンサイ（サトウダイコン）などが挙げられる。前記タデ科植物としては、ソバなどが挙げられる。 Examples of the cruciferous plant include Arabidopsis, Chinese cabbage, rapeseed, cabbage, cauliflower, and Japanese radish. Examples of the legumes include soybean, adzuki bean, kidney bean, pea, cowpea, alfalfa and the like. Examples of the Solanaceae plant include tomato, eggplant, potato, tobacco, and red pepper. Examples of the Cucurbitaceae plant include makuwari, cucumber, melon and watermelon. Examples of the bindweed plant include morning glory, sweet potato (sweet potato), bindweed, and the like. Examples of the Rosaceae plant include roses, strawberries, apples and the like. Examples of the mulberry plant include mulberry, fig, and rubber tree. Examples of the mallow family plants include cotton and kenaf. Examples of the Asteraceae plant include lettuce and the like. Examples of the Amaranthaceae plant include sugar beet (sugar radish). Examples of the Polygonaceae plant include buckwheat.
　前記裸子植物としては、マツ、スギ、イチョウ及びソテツなどが挙げられる。 Examples of the gymnosperms include pine, cedar, ginkgo and cycad.
（タンパク質の金粒子への結合を促進する方法）
　本発明のタンパク質の金粒子への結合を促進する方法は、ＭｇＣｌ２、及びＤＴＴを除く塩濃度の終濃度が、０より多く３．５ｍＭ以下である緩衝液、又は塩を含まない緩衝液を用いて、タンパク質の金粒子への結合を促進する方法である。
　前記緩衝液を用いて、タンパク質の金粒子への結合を促進する方法は、前述の緩衝液を用いて、金粒子をタンパク質によりコーティングする方法のとおりである。 (Method of promoting binding of protein to gold particles)
The method of promoting the binding of the protein to the gold particles of the present invention is performed by using a buffer solution in which the final concentration of salt concentration excluding MgCl 2 and DTT is more than 0 and 3.5 mM or less, or a buffer solution containing no salt. It is a method of promoting the binding of proteins to gold particles.
The method of promoting the binding of the protein to the gold particles using the buffer solution is the same as the method of coating the gold particles with the protein using the buffer solution described above.
　塩濃度を下げることで、効率よくタンパク質を被覆した金粒子をマクロキャリア上にスポットして遺伝子銃により生物に撃ち込むことにより、例えば、ゲノム編集の効率を上げられるなどの効果が得られる。
　以下に実施例を用いて本発明を説明するが、本発明は何らこれら実施例により限定されるものではない。 By lowering the salt concentration, gold particles coated with protein can be efficiently spotted on the macrocarrier and shot into an organism with a gene gun, whereby effects such as an increase in the efficiency of genome editing can be obtained.
The present invention will be described below with reference to examples, but the present invention is not limited to these examples.
　以下に金粒子にタンパク質を結合させるために用いた各種ｂｕｆｆｅｒの名前と組成を示す。 The names and compositions of the various buffers used to bind the protein to the gold particles are shown below.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
Ｂｕｆｆｅｒ組成(塩濃度)の影響
方法
（１）１．５ｍＬチューブに３μｇ　ＳｐＣａｓ９（ＴａＫａＲａ）、１μｇ　ｓｇＲＮＡ、Ｎｕｃｌｅａｓｅ－ｆｒｅｅ　ｗａｔｅｒ　３．２５μＬ、×１０　各種ｂｕｆｆｅｒ　１μＬを加え、ピペッティングにより、よく混ぜる。
（２）３６０μｇ　金粒子(直径０．６μｍ)を添加し、ピペッティングにより混ぜ、１０分間静置(最終ｖｏｌｕｍｅ　１０μＬ)する。
（３）遠心後、上清を新しいエッペンチューブへ移し、×２　ＳＤＳ－ＰＡＧＥ用ｓａｍｐｌｅ　ｂｕｆｆｅｒ　１０μＬを加え、９５℃３分間加熱する。
（４）ＳＤＳ－ＰＡＧＥによりタンパク質(Ｃａｓ９)を分離後、ＣＢＢ染色する。
（５）各処理区において、Ｃａｓ９タンパク質由来のバンドを定量化する。コントロール(金粒子なし)のバンドと比較し、金粒子に吸着したと考えられるＣａｓ９タンパク質量を算出する。
　その結果を、図１～図４に示す。 Method of influencing Buffer composition (salt concentration) (1) Add 3 μg SpCas9 (TaKaRa), 1 μg sgRNA, Nuclease-free water 3.25 μL, and 10 μl various buffer 1 μL to a 1.5 mL tube, and mix well by pipetting.
(2) Add 360 μg of gold particles (diameter 0.6 μm), mix by pipetting, and let stand for 10 minutes (final volume 10 μL).
(3) After centrifugation, the supernatant is transferred to a new Eppendorf tube, 10 μL of sample buffer for ×2 SDS-PAGE is added, and the mixture is heated at 95° C. for 3 minutes.
(4) After protein (Cas9) is separated by SDS-PAGE, CBB staining is performed.
(5) In each treatment group, the band derived from Cas9 protein is quantified. The amount of Cas9 protein considered to be adsorbed to the gold particles is calculated by comparing with the band of the control (without gold particles).
The results are shown in FIGS.
　図１は、各種ｂｕｆｆｅｒを用いた際のタンパク質の金粒子への結合量（％）をプロットしたものである。図１に示すように、Ａが最も金粒子への結合量が多かった。Ａは、ＮａＣｌ濃度が０であるので（表１参照）、塩濃度が低いほど、タンパク質の金粒子への結合量は多くなると考えられた。 Fig. 1 is a plot of the binding amount (%) of the protein to the gold particles when various buffers were used. As shown in FIG. 1, A had the largest amount of binding to the gold particles. Since A had a NaCl concentration of 0 (see Table 1), it was considered that the lower the salt concentration, the greater the amount of protein bound to the gold particles.
　図２は、終濃度１．０ｍＭ　ＴｒｉｓＨＣｌ（ｐＨ７．５）において、ＮａＣｌ濃度を変化させた際の金粒子へのタンパク質の結合量を示したものである。図２に示すように、Ｔｒｉｓ濃度が一定の場合、ＮａＣｌ濃度が上がるほど、金粒子へのタンパク質の結合量が減少しているのがわかる。 Fig. 2 shows the amount of protein bound to gold particles when the NaCl concentration was changed at a final concentration of 1.0 mM TrisHCl (pH 7.5). As shown in FIG. 2, it can be seen that when the Tris concentration is constant, the binding amount of protein to the gold particles decreases as the NaCl concentration increases.
　図３は、Ｔｒｉｓ濃度の、タンパク質の金粒子への結合量に対する影響を調べたものであるが、ＮａＣｌの終濃度が１．０ｍＭの場合、Ｔｒｉｓ濃度が増加するに連れて、金粒子へのタンパク質結合量が減少していることがわかる。 FIG. 3 shows the effect of the Tris concentration on the amount of protein bound to the gold particles. When the final concentration of NaCl was 1.0 mM, the Tris concentration increased as the Tris concentration increased. It can be seen that the amount of protein bound is reduced.
　図４は、ＭｇＣｌ２の、金粒子へのタンパク質の結合量に及ぼす影響を調べたものであるが、ＭｇＣｌ２は特にタンパク質の金粒子への結合量には影響はないと考えられた。 Figure 4 is a MgCl 2, but in which was studied the effect on binding of protein to the gold particles, MgCl 2 is was not considered particularly affect the binding amount of the gold particles of the protein.
　本発明の態様としては、例えば、以下のものなどが挙げられる。
　＜１＞　以下のいずれかの緩衝液を用いることを特徴とする、金粒子をタンパク質によりコーティングする方法である：１）前記緩衝液における、ＭｇＣｌ２、及びＤＴＴを除く塩濃度の終濃度が、０より多く３．５ｍＭ以下であるる緩衝液。２）塩を含まない緩衝液。
　＜２＞　タンパク質がコーティングされた金粒子の製造方法であって、以下のいずれかの緩衝液を用いて、金粒子をタンパク質によりコーティングするコーティング工程を有することを特徴とする方法である：１）前記緩衝液における、ＭｇＣｌ２、及びＤＴＴを除く塩濃度の終濃度が、０より多く３．５ｍＭ以下である緩衝液。２）塩を含まない緩衝液。
　＜３＞　金粒子をスポットしたマクロキャリアの製造方法であって、以下のいずれかの緩衝液を用いて、金粒子をタンパク質によりコーティングするコーティング工程、及び、前記コーティング工程で得られた金粒子をマクロキャリアにスポットするスポット工程を含むことを特徴とする方法である：１）前記緩衝液における、ＭｇＣｌ２、及びＤＴＴを除く塩濃度の終濃度が、０より多く３．５ｍＭ以下である緩衝液。２）塩を含まない緩衝液。
　＜４＞　前記＜２＞に記載の方法で製造した金粒子、または前記＜３＞に記載の方法で製造したマクロキャリアを用いて遺伝子銃により細胞にタンパク質を導入する方法である。
　＜５＞　前記緩衝液が、さらに両親媒性分子と塩基性タンパク質を含む、前記＜１＞～＜４＞のいずれかに記載の方法である。
　＜６＞　前記緩衝液が、さらに核酸を含む、前記＜１＞～＜５＞のいずれかに記載の方法である。
　＜７＞　前記両親媒性分子と塩基性タンパク質が１，４－ビス（３－オレオイルアミドプロピル）ピペラジンおよびヒストンＨ１である、前記＜５＞または＜６＞に記載の方法である。
　＜８＞　前記＜４＞～＜７＞のいずれかに記載の方法を用いてゲノム編集を行う方法である。
　＜９＞　前記＜４＞～＜８＞のいずれかに記載の方法により得られる細胞、組織、器官、生物個体ならびに／またはその後代および／もしくは種子である。
　＜１０＞　以下のいずれかの緩衝液を用いて、タンパク質の金粒子への結合を促進する方法である：１）前記緩衝液における、ＭｇＣｌ２、及びＤＴＴを除く塩濃度の終濃度が、０より多く３．５ｍＭ以下である緩衝液。２）塩を含まない緩衝液。 Examples of aspects of the present invention include the following.
<1> A method for coating gold particles with a protein, which comprises using one of the following buffers: 1) The final concentration of salt concentration excluding MgCl 2 and DTT in the buffer is A buffer solution that is greater than 0 and less than or equal to 3.5 mM. 2) Salt-free buffer.
<2> A method for producing protein-coated gold particles, which comprises a coating step of coating gold particles with protein using one of the following buffers: 1) A buffer solution in which the final concentration of the salt concentration excluding MgCl 2 and DTT in the buffer solution is more than 0 and 3.5 mM or less. 2) Salt-free buffer.
<3> A method for producing a macro carrier in which gold particles are spotted, comprising a coating step of coating gold particles with a protein using one of the following buffer solutions, and the gold particles obtained in the coating step. The method is characterized by comprising a spotting step of spotting on a macrocarrier: 1) A buffer solution in which the final salt concentration excluding MgCl 2 and DTT in the buffer solution is more than 0 and 3.5 mM or less. .. 2) Salt-free buffer.
<4> A method of introducing a protein into a cell by a gene gun using the gold particles produced by the method described in <2> or the macrocarrier produced by the method described in <3>.
<5> The method according to any one of <1> to <4>, wherein the buffer solution further contains an amphipathic molecule and a basic protein.
<6> The method according to any one of <1> to <5>, wherein the buffer solution further contains a nucleic acid.
<7> The method according to <5> or <6>, wherein the amphipathic molecule and the basic protein are 1,4-bis(3-oleoylamidopropyl)piperazine and histone H1.
<8> A method for genome editing using the method according to any one of <4> to <7>.
<9> A cell, tissue, organ, individual organism and/or progeny and/or seed obtained by the method according to any one of <4> to <8>.
<10> A method of promoting the binding of a protein to gold particles using any of the following buffers: 1) The final concentration of salt concentration excluding MgCl 2 and DTT in the buffer is 0 A buffer that is more than 3.5 mM. 2) Salt-free buffer.
　本発明は、バイオテクノロジー産業、農業、新品種育種産業等において利用できる。 The present invention can be used in the biotechnology industry, agriculture, breed breeding industry, etc.

Claims (10)

  1. 　以下のいずれかの緩衝液を用いることを特徴とする、金粒子をタンパク質によりコーティングする方法：
    　１）前記緩衝液における、ＭｇＣｌ２、及びＤＴＴを除く塩濃度の終濃度が、０より多く３．５ｍＭ以下である緩衝液。
    　２）塩を含まない緩衝液。     Method for coating gold particles with a protein, characterized by using one of the following buffers:
    1) A buffer solution in which the final salt concentration excluding MgCl 2 and DTT in the buffer solution is more than 0 and 3.5 mM or less.
    2) Salt-free buffer.
  2. 　タンパク質がコーティングされた金粒子の製造方法であって、
    　以下のいずれかの緩衝液を用いて、金粒子をタンパク質によりコーティングするコーティング工程を有することを特徴とする方法：
    　１）前記緩衝液における、ＭｇＣｌ２、及びＤＴＴを除く塩濃度の終濃度が、０より多く３．５ｍＭ以下である緩衝液。
    　２）塩を含まない緩衝液。     A method for producing protein-coated gold particles, comprising:
    A method comprising the step of coating gold particles with a protein using any of the following buffers:
    1) A buffer solution in which the final salt concentration excluding MgCl 2 and DTT in the buffer solution is more than 0 and 3.5 mM or less.
    2) Salt-free buffer.
  3. 　金粒子をスポットしたマクロキャリアの製造方法であって、
    　以下のいずれかの緩衝液を用いて、金粒子をタンパク質によりコーティングするコーティング工程、及び、
    　前記コーティング工程で得られた金粒子をマクロキャリアにスポットするスポット工程を含むことを特徴とする方法：
    　１）前記緩衝液における、ＭｇＣｌ２、及びＤＴＴを除く塩濃度の終濃度が、０より多く３．５ｍＭ以下である緩衝液。
    　２）塩を含まない緩衝液。     A method of manufacturing a macro carrier in which gold particles are spotted,
    A coating step of coating gold particles with a protein using one of the following buffers, and
    A method comprising a spotting step of spotting the gold particles obtained in the coating step on a macrocarrier:
    1) A buffer solution in which the final salt concentration excluding MgCl 2 and DTT in the buffer solution is more than 0 and 3.5 mM or less.
    2) Salt-free buffer.
  4. 　請求項２に記載の方法で製造した金粒子、または請求項３に記載の方法で製造したマクロキャリアを用いて遺伝子銃により細胞にタンパク質を導入する方法。 A method of introducing a protein into a cell by a gene gun using the gold particle produced by the method of claim 2 or the macrocarrier produced by the method of claim 3.
  5. 　前記緩衝液が、さらに両親媒性分子と塩基性タンパク質を含む、請求項１～４のいずれかに記載の方法。 The method according to any one of claims 1 to 4, wherein the buffer solution further contains an amphipathic molecule and a basic protein.
  6. 　前記緩衝液が、さらに核酸を含む、請求項１～５のいずれかに記載の方法。 The method according to any one of claims 1 to 5, wherein the buffer solution further contains a nucleic acid.
  7. 　前記両親媒性分子と塩基性タンパク質が１，４－ビス（３－オレオイルアミドプロピル）ピペラジンおよびヒストンＨ１である、請求項５または６に記載の方法。 The method according to claim 5 or 6, wherein the amphipathic molecule and the basic protein are 1,4-bis(3-oleoylamidopropyl)piperazine and histone H1.
  8. 　請求項４～７のいずれかに記載の方法を用いてゲノム編集を行う方法。 A method for editing a genome using the method according to any one of claims 4 to 7.
  9. 　請求項４～８のいずれかに記載の方法により得られる細胞、組織、器官、生物個体ならびに／またはその後代および／もしくは種子。 A cell, tissue, organ, individual organism and/or a progeny and/or seed obtained by the method according to any one of claims 4 to 8.
  10. 　以下のいずれかの緩衝液を用いて、タンパク質の金粒子への結合を促進する方法：
    　１）前記緩衝液における、ＭｇＣｌ２、及びＤＴＴを除く塩濃度の終濃度が、０より多く３．５ｍＭ以下である緩衝液。
    　２）塩を含まない緩衝液。     Methods of promoting protein binding to gold particles using any of the following buffers:
    1) A buffer solution in which the final salt concentration excluding MgCl 2 and DTT in the buffer solution is more than 0 and 3.5 mM or less.
    2) Salt-free buffer.
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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001337065A (en) * 2000-05-24 2001-12-07 Bio Focus Ltd Electrochemical membrane strip biosensor
JP2004532850A (en) * 2001-04-30 2004-10-28 サイティミュン サイエンス， インコーポレイテッド Colloidal metal compositions and methods
JP2009516199A (en) * 2005-11-18 2009-04-16 アメリカ合衆国 Modified cardiolipin and uses thereof
WO2017150733A1 (en) * 2016-03-04 2017-09-08 田中貴金属工業株式会社 Immunochromatography device
WO2017195906A1 (en) * 2016-05-13 2017-11-16 株式会社カネカ Plant genome editing method
US20180186842A1 (en) * 2016-12-30 2018-07-05 Synthetic Genomics, Inc. High productivity algal mutants having reduced photosynthetic antenna
CN108498460A (en) * 2017-02-24 2018-09-07 国家纳米科学中心 Gold nanoclusters-liposome composite particles and its preparation method and application

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001337065A (en) * 2000-05-24 2001-12-07 Bio Focus Ltd Electrochemical membrane strip biosensor
JP2004532850A (en) * 2001-04-30 2004-10-28 サイティミュン サイエンス， インコーポレイテッド Colloidal metal compositions and methods
JP2009516199A (en) * 2005-11-18 2009-04-16 アメリカ合衆国 Modified cardiolipin and uses thereof
WO2017150733A1 (en) * 2016-03-04 2017-09-08 田中貴金属工業株式会社 Immunochromatography device
WO2017195906A1 (en) * 2016-05-13 2017-11-16 株式会社カネカ Plant genome editing method
US20180186842A1 (en) * 2016-12-30 2018-07-05 Synthetic Genomics, Inc. High productivity algal mutants having reduced photosynthetic antenna
CN108498460A (en) * 2017-02-24 2018-09-07 国家纳米科学中心 Gold nanoclusters-liposome composite particles and its preparation method and application

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MIZUTA, YOKO ET AL.: "Genome editing of pollen", RESEARCH PRESENTATION RECORD OF THE 82ND ANNUAL MEETING OF THE BOTANICAL SOCIETY OF JAPAN, vol. 82, September 2018 (2018-09-01), pages 256 *
THOBHANI, S. ET AL.: "Bioconjugation and characterisation of gold colloid-labelled proteins", J. IMMUNOL. METHODS, vol. 356, no. 1-2, 2010, pages 60 - 69, XP027015644 *

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