JP7526396B2 - A method for producing a carrier carrying fine particles coated with a substance to be introduced into cells, which is used in particle bombardment method - Google Patents

Description

The present invention relates to a method for producing a carrier used in particle bombardment method, which carries microparticles coated with a substance to be introduced into cells. The present invention also relates to the carrier and a method for introducing a substance into cells by particle bombardment method using the carrier.

Introducing substances such as nucleotides and peptides into cells is extremely meaningful not only for basic research but also for various industrial applications. However, it is difficult to introduce these substances into cells while maintaining their activity. In particular, plants have hard cell walls, which makes it extremely difficult to introduce substances from the outside compared to animals.

Methods for introducing substances into plant cells include chemical methods (polyethylene glycol method) and physical methods (electroporation method, particle bombardment method). Of these, particle bombardment method is a method in which fine particles made of gold or tungsten, etc., coated with DNA or proteins, are fired at high speed as bullets to introduce substances into cells. This method is simple and applicable to a wide range of plants, so it is commonly used as a method for introducing substances into many tissues, cells, and plant cells (Non-Patent Documents 1-3 and Patent Document 1). Moreover, it has been reported that this method is particularly effective for mature pollen with hard cell walls in various angiosperms (Non-Patent Document 4).

In addition, with the advent of genome editing technology in recent years, particle bombardment method has been attracting attention as a technology that can easily perform plant transformation.For example, when the complex of Cas9 protein and guide RNA is introduced by particle bombardment method for the purpose of genome editing, Cas9 protein and guide RNA are mixed in Cas9 reaction buffer (20 mM HEPES, pH 7.5, 150 mM KCl, 10 mM MgCl ₂ , 0.5 mM DTT), and then gold fine particles are mixed to coat the fine particles with the complex, and the mixture is applied to a carrier (carrier) and air-dried to support the fine particles on the carrier (Non-Patent Document 3).

US Patent Application Publication No. 2014/96284

J. Finer, J. et al., Curr Top Microbiol Immunol., 1999, vol. 240, pp. 59-80 Martin-Ortigosa S. et al., Transgenic Res. 2014, Vol. 23, No. 5, pp. 743-756 Liang Z. et al., Nat Commun., 2017, 8:14261 Schreiber, D. N. et al., Plant Molecular Biology Reporter, 2003, vol. 1, pp. 31-41

The present invention aims to provide a method for producing a carrier used in particle bombardment, in which a substance (nucleotide-peptide complex) to be introduced into cells is coated on microparticles while maintaining its activity, and the microparticles are stably supported.

As mentioned above, particle bombardment is generally known as a technique for introducing DNA into cells and tissues. In particular, with the advent of genome editing technology in recent years, it has attracted attention as a technique that can easily transform plants.

However, when a carrier carrying microparticles coated with a complex of the Cas9 protein, a genome editing system, and the guide RNA is prepared by using such a conventional method (the method described in Non-Patent Document 3), the present inventors have found for the first time that inorganic salts (KCl) precipitate during the drying process of the carrier, and the layer of microparticles carried on the surface of the carrier becomes fragile, as shown in Reference Examples 3 and 4 described below. Furthermore, many of the precipitates were 100 μm or larger, and gold particles were unevenly present on the surface of the carrier in the areas other than the precipitates. Since the size of a cell is usually 100 μm or less, it is not only impossible to introduce a substance into the cell using the precipitates, but it is estimated that the impact of the precipitates may cause damage to the cell. On the other hand, if the inorganic salts are removed from the Cas9 reaction buffer, it becomes difficult to dissolve substances such as the Cas9 protein and the guide RNA, and there is a concern that the activity of the substances may decrease.

Furthermore, as shown in Reference Example 3 below, the inventors also found that drying by air drying reduces the activity of the Cas9 protein-guide RNA complex.

Therefore, in order to solve the problems in the conventional technology that were first revealed as described above, that is, to produce a carrier in which a nucleotide-peptide complex such as a Cas9 protein-guide RNA complex covers a microparticle while maintaining its activity and the microparticle is stably supported, the inventors have conducted extensive research. As a result, they first came up with the idea of first dissolving the nucleotide and peptide in a neutral buffer solution containing inorganic salts and/or sugars, and then minimizing the content of inorganic salts and sugars that significantly affect the precipitation.

In fact, the inorganic salts and sugars were removed from the solution containing the Cas9 protein-guide RNA complex by buffer exchange, and it was found that the activity of the complex was maintained. Furthermore, as shown in Reference Example 3 below, drying by air drying or freeze-drying has problems with maintaining the activity of the Cas9 protein-guide RNA complex and the stability of the carrier, but it was found that degassing can solve these problems, leading to the completion of the present invention.

That is, the present invention relates to a method for producing a carrier carrying fine particles coated with a substance to be introduced into cells, which is used in particle bombardment, and more specifically, provides the method described below.
<1> A method for producing a carrier carrying fine particles coated with a substance, which is used in a particle bombardment method, comprising the steps of:
The substance is a complex of a peptide and a nucleotide, which is introduced into a cell by a particle bombardment method, and the method includes the following steps (1) to (5): (1) dissolving the peptide and the nucleotide in a neutral buffer containing an inorganic salt and/or a sugar;
(2) removing the inorganic salts and the sugars from the lysate obtained in step (1) by buffer exchange;
(3) mixing the solution obtained in step (2) with the microparticles;
(4) applying the mixture obtained in step (3) onto the surface of a carrier;
(5) A step of drying the carrier coated with the mixed solution in the step (4) by a degassing drying method.
<2> The method according to <1>, wherein the inorganic salt is a potassium salt and the sugar is sucrose.
<3> The method according to <1> or <2>, wherein the neutral buffer solution is a Tris buffer solution having a pH of 7 to 8 or a HEPES buffer solution having a pH of 7 to 8.
<4> The method according to any one of <1> to <3>, wherein the peptide is a Cas9 protein and the nucleotide is a guide RNA.
<5> A carrier carrying fine particles coated with a substance, which is used in a particle bombardment method,
The substance is a peptide-nucleotide complex that is introduced into cells by particle bombardment, and
A carrier, wherein on the surface on which the fine particles are supported, precipitates in an area within 10 mm from the center are less than 100 μm in size.
<6> A carrier carrying fine particles coated with a substance, the carrier being produced by the method according to any one of <1> to <4>.
<7> A method for introducing a substance into a cell by particle bombardment, comprising:
A method comprising the step of applying pressure to the carrier according to <5> or <6>, thereby shooting the microparticles released from the carrier into the cells.
<8> A kit for use in the method according to any one of <1> to <4>, comprising at least one substance selected from the group consisting of substances described in the following (A) to (F): (A) the peptide and the nucleotide; (B) a neutral buffer solution containing an inorganic salt and/or a sugar; (C) an ultrafiltration membrane, a dialysis membrane, or a chromatography immobilized phase used for the buffer exchange; (D) a solution used for performing the buffer exchange with the lysate obtained in the step (1); (E) the fine particles; and (F) a carrier on which the mixed solution is applied.

According to the present invention, it is possible to provide a method for producing a carrier used in particle bombardment, in which a substance (nucleotide-peptide complex) to be introduced into cells covers the microparticles while maintaining its activity, and the microparticles are stably supported. Furthermore, since the microparticles can be stably supported, it is possible to produce and provide the carrier, which can also be transported and stored. In addition, the precipitation of salt on the carrier surface is reduced, and it is possible to introduce the complex into cells and make it function while suppressing damage caused by the precipitation.

FIG. 1 is a diagram showing an outline of the particle bombardment method of the present invention (before a substance is bombarded into a cell). FIG. 1 is a diagram showing an outline of the particle bombardment method of the present invention (when a substance is bombarded into a cell). This is a diagram showing the results of evaluating the cleavage activity of the Cas9 protein or Cas9 protein-guide RNA complex prepared under conditions 1 to 6 shown in Reference Example 2. In the figure, the upper part is a gel electrophoresis photograph showing the results of detecting the cleavage status of the template DNA by staining with SYBR safe, the triangle indicates the position of the template DNA cleaved by the Cas9 protein, and the number (%) indicates the cleavage rate. The lower part is a photograph of SDS-PAGE showing the results of detecting the Cas9 protein by CBB staining. In addition, "M" in each photograph indicates the result of electrophoresis of a DNA or protein marker. This figure shows the results of evaluating the cleavage activity of Cas9 protein-guide RNA complexes prepared under conditions 1 to 4 shown in Reference Example 3 (the notations in the figure are the same as those in FIG. 2). This is a photograph showing the results of observing a disk carrying a Cas9 protein-guide RNA complex in a particle bombardment method, prepared using condition 1 shown in Reference Example 3. In the figure, the central region shown in "(a)" is the "substance-coated microparticle" according to the present invention, which is circularly coated in the center of the carrier, and the white precipitate in the central portion is shown by a triangle in the figure. This is a photograph showing the results of observing a disk carrying a Cas9 protein-guide RNA complex in a particle bombardment method, prepared under the conditions described in Example 1. In the figure, the central region indicated by "(a)" is the "substance-coated microparticle" according to the present invention, which is circularly coated at the center of the carrier. This figure shows the results of evaluating the cleavage activity of the Cas9 protein-guide RNA complex-supported disk in the particle bombardment method ("1" in the figure) prepared under the conditions described in Example 1, and the Cas9 protein-guide RNA complex-supported disk in the particle bombardment method ("2" in the figure) prepared under the conditions described in Example 1. "3" and "4" in the figure show the results of electrophoresis of only template DNA and only Cas9 protein, respectively. The notations in the figure are the same as those in FIG. 2, but the numbers (%) shown at the bottom indicate the cleavage efficiency. This is a photograph showing the results of observing, with an optical microscope, a disk carrying the Cas9 protein-guide RNA complex prepared by the particle bombardment method under the conditions described in Example 1. This is a photograph showing the results of observing, with an optical microscope, a disk carrying a Cas9 protein-guide RNA complex produced by the particle bombardment method under the conditions described in Non-Patent Document 3.

<Production Method of the Present Invention>
As shown in the Examples below, by taking the following steps (1) to (5), it is possible to provide a method for producing a carrier used in particle bombardment, in which a substance (nucleotide-peptide complex) to be introduced into cells covers microparticles while maintaining its activity, and the microparticles are stably supported.

Thus, the present invention provides
the substance is a peptide-nucleotide complex that is introduced into cells by particle bombardment;
(1) dissolving the peptide and the nucleotide in a neutral buffer containing an inorganic salt and/or a sugar;
(2) removing the inorganic salts and the sugars from the lysate obtained in step (1) by buffer exchange;
(3) mixing the solution obtained in step (2) with the microparticles;
(4) applying the mixture obtained in step (3) onto the surface of a carrier;
(5) Provided is a method for producing a carrier carrying fine particles coated with a substance in a particle bombardment method, the method including a step of drying the carrier coated with the mixed liquid in step (4) by a degassing and drying method.

In the manufacturing method of the present invention, first, the peptide and the nucleotide that constitute the complex to be introduced into the cell are dissolved in a neutral buffer solution containing inorganic salts and/or sugars.

(Substances to be introduced into cells)
In the present invention, the substance introduced into cells by particle bombardment is a complex of peptide and nucleotide. The "peptide" according to the present invention includes oligopeptides, polypeptides, and proteins, and the nucleotides (DNA, RNA, etc.) include oligonucleotides, polynucleotides, and nucleic acids. There is no particular limitation on the complexes formed by such nucleotides and peptides, and examples thereof include RNA-protein complexes and DNA-protein complexes, and more specifically, complexes of Cas proteins and guide RNAs, ribosomes, spliceosomes, and chromatin. Note that at least one of these substances may be bound to a labeling substance. Examples of such labeling substances include fluorescent compounds (fluorescein, FITC, indocyanine green, fluorescence-emitting metals ( ¹⁵² Eu, lanthanum series, etc.), IRDye800 series), marker proteins (for example, fluorescent proteins such as GFP, luminescent enzyme proteins such as luciferase), and tag proteins (His tag peptides, GST proteins, etc.). In addition, a protease recognition site may be provided between the labeling substance and the peptide or nucleotide, so that they can be separated by cleavage with the protease.

In the present invention, the "Cas protein" is a CRISPER (clustered regularly interspaced short palindromic repeat, CRISPER)-associated enzyme (nuclease), which may be a class 1 CRISPER-associated enzyme (e.g., type I such as Cas3, type IV, type III such as Cas10) or a class 2 CRISPER-associated enzyme (e.g., type II such as Cas9, type V such as Cas12a (Cpf1), Cas12b (C2c1), Cas12e (CasX) and Cas14, type VI such as Cas13), but is preferably a class 2 CRISPER-associated enzyme, more preferably a type II CRISPER-associated enzyme, and even more preferably Cas9. In addition, typical amino acid sequences and nucleotide sequences of Cas proteins are registered in public databases, for example, GenBank (http://www.ncbi.nlm.nih.gov), and these can be used in the present invention.

In the present invention, the "Cas9 protein" may be any protein that can be used in the CRISPR/Cas9 system, and binds to a guide RNA to form a complex, and is targeted to a target DNA region to cleave the target double-stranded DNA. Cas9 proteins of various origins are known, and those exemplified in WO2014/131833 can be used. Preferably, the Cas9 protein (SpCas9) derived from Streptococcus pyogenes is used. The amino acid sequence and nucleotide sequence of the Cas9 protein are registered in a public database, for example, GenBank (http://www.ncbi.nlm.nih.gov) (for example, UniProtKB/Swiss-Prot: Q99ZW2), and these can be used in the present invention.

In addition to the naturally occurring amino acid sequences registered in the database, the Cas9 protein of the present invention may be a mutant containing an amino acid sequence in which one or more amino acids have been deleted, substituted, added, or inserted relative to the naturally occurring amino acid sequence. Here, "multiple" means 1 to 50, preferably 1 to 30, and more preferably 1 to 10. Furthermore, the Cas9 protein of the present invention includes an amino acid sequence having 80% or more, more preferably 90% or more, even more preferably 95% or more, and most preferably 99% or more sequence identity with the naturally occurring amino acid sequence, or a polypeptide consisting of said amino acid sequence, so long as the activity of the original protein is maintained. Comparison of amino acid sequences can be performed by known methods, such as BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) or the like, for example, using default settings.

An example of such a mutant is the Cas9 protein (nCas protein), which has a mutation introduced into one of the catalytic sites and exhibits nickase activity. By using the nCas protein, off-target effects can be reduced. In addition, the addition of a nuclear localization signal promotes localization to the nucleus within the cell, resulting in efficient DNA editing.

The "guide RNA" of the present invention is an RNA that contains a nucleotide sequence that interacts with the Cas9 protein and a nucleotide sequence (targeting nucleotide sequence) that is complementary to the sequence of the target DNA region. In the present invention, the "guide RNA" may be a single-molecule guide RNA that contains a crRNA and a tracrRNA, or a dual-molecule guide RNA (dual-RNA) that consists of a crRNA fragment and a tracrRNA fragment.

The targeting nucleotide sequence in the crRNA is usually a base sequence consisting of 12 to 50 nucleotides, preferably 17 to 30 nucleotides, more preferably 17 to 25 nucleotides, and is selected to target a region adjacent to the PAM (proto-spacer adjacent motif) sequence.

The crRNA further contains a nucleotide sequence on the 3' side that can interact (hybridize) with the tracrRNA. On the other hand, the tracrRNA contains a nucleotide sequence on the 5' side that can interact (hybridize) with a nucleotide sequence of a portion of the crRNA. The double-stranded RNA formed by the interaction of these nucleotide sequences interacts with the Cas9 protein.

(Neutral buffer containing inorganic salts and/or sugars)
The buffer solution for dissolving the above-mentioned peptides and nucleotides to form a complex of these substances has a neutral pH range (for example, pH 6.5 to 8.0, preferably pH 7.0 to 8.0, more preferably pH 7.5) in order to maintain the activity of the complex. The substance having a buffering effect (buffering agent) contained in the buffer solution to set the pH of the buffer solution in the above range is not particularly limited as long as it has the said effect, and examples thereof include Tris (Tris-HCl), HEPES (HEPES-NaOH), phosphoric acid (NaH ₂ PO ₄ -Na ₂ HPO ₄ ), and MOPS (MOPS-NaOH). Of these buffering agents, Tris or HEPES is preferably used in the present invention when introducing the buffering agent into plants, particularly pollen cells. The concentration of the buffer is not particularly limited as long as the pH range is neutral, but is preferably 2 to 200 mM, more preferably 5 to 100 mM, further preferably 10 to 50 mM, and particularly preferably 20 mM.

The "buffer solution" according to the present invention further contains an inorganic salt and/or a sugar to dissolve the nucleotide-peptide complex and maintain its activity. Examples of inorganic salts include potassium salts (potassium chloride, potassium phosphate, potassium nitrate, potassium hydroxide, etc.), sodium salts (sodium chloride, sodium acetate, sodium bicarbonate, sodium nitrate, etc.), calcium salts (calcium chloride, calcium sulfate, calcium acetate, etc.), magnesium salts (magnesium chloride, magnesium sulfate, etc.), and ammonium salts (ammonium carbonate, etc.). For introduction into plants, particularly pollen cells, potassium salts are preferred. Examples of sugars include sucrose, fructose, lactose, mannitol, glycerol, polyethylene glycol (PEG), trehalose, glycine, glucose, dextran, erythritol, and maltose. From the viewpoint of maintaining the stability of the nucleotide-peptide complex while being easily dried, trehalose and sucrose are preferred, and sucrose is more preferred.

The buffer solution of the present invention may contain at least one of these compounds (for example, only potassium chloride or only sucrose), but from the viewpoint of maintaining the stability of the nucleotide-peptide complex while making it easier to dry, it is preferable that the buffer solution contains a potassium salt and sucrose. There are no particular limitations on the content of inorganic salts or sugars in the buffer solution, but from the viewpoint of better dissolving the nucleotide-peptide complex and easier suppression of precipitation of the complex, the content of inorganic salts is preferably 100 to 500 mM, more preferably 200 to 300 mM, and the content of sugars is preferably 0.5 to 5%, or 1 to 2%.

The buffer solution of the present invention may contain other substances in addition to the buffering agent, inorganic salts, and sugars. Examples of such other substances include reducing agents (DTT, etc.) and protease inhibitors (PSDF, etc.).

Furthermore, the dissolved concentrations of the peptide and nucleotide in the buffer solution according to the present invention are not particularly limited, but from the viewpoint of facilitating the formation of a nucleotide-peptide complex and facilitating the introduction of the complex into cells in an amount suitable for the cell, the peptide concentration is preferably 10 to 1000 ng/μl, more preferably 50 to 500 ng/μl, and even more preferably 100 to 200 ng/μl, and the nucleotide concentration is preferably 1 to 100 ng/μl, more preferably 10 to 50 ng/μl, and even more preferably 20 to 30 ng/μl.

(Removal of inorganic salts and sugars)
In the present invention, the inorganic salts and sugars are removed from the solution of the above-mentioned nucleotide-peptide complex by buffer exchange, thereby suppressing the precipitation of these inorganic salts and sugars on the surface of the carrier described below, and making it possible to stably support the microparticles described below.

In the present invention, "buffer exchange" means partially or completely exchanging inorganic salts, sugars, buffers, etc. in a solution containing a nucleotide-peptide complex with those of another solution (hereinafter also referred to as a "buffer exchange solution"), thereby reducing the concentration of the inorganic salts and/or sugars in the solution containing the complex while leaving the nucleotide-peptide complex. Furthermore, "removal" of the inorganic salts and sugars from the solution of the nucleotide-peptide complex means making the solution substantially free of the inorganic salts and sugars. The state in which the inorganic salts are present is preferably 0.1 mM or less, more preferably 0.01 mM or less, and particularly preferably 0 mM, in the solution of the nucleotide-peptide complex. The state in which the sugars are present is preferably 0.01% or less, even more preferably 0.001% or less, and particularly preferably 0% in the solution of the nucleotide-peptide complex.

As a method for "buffer exchange," a known desalting and/or buffer exchange method can be used. Examples of such methods include ultrafiltration, dialysis, and chromatography.

The "ultrafiltration" according to the present invention can be carried out using an ultrafiltration membrane with a molecular weight cutoff that does not allow nucleotide-peptide complexes to pass through, but allows inorganic salts, sugars, and other low molecular weight molecules to pass through. Those skilled in the art can select the separation performance of the ultrafiltration membrane appropriately, taking into consideration the molecular weights of the nucleotides and peptides to be treated, but the molecular weight cutoff is preferably 5 to 150 kDa, more preferably 10 to 100 kDa, even more preferably 20 to 70 kDa, and particularly preferably 50 kDa.

Examples of ultrafiltration membranes include cellulose-based, polysulfone-based, polyacrylonitrile-based, and polyfluorohydrocarbon-based polymer membranes. Examples of cellulose-based polymer membranes include regenerated cellulose, cellulose acetate, nitrocellulose, and cellulose mixed esters. Examples of polysulfone-based polymer membranes include polysulfone, polyethersulfone, polyphenylsulfone, and polyarylsulfone. Examples of polyfluorohydrocarbon-based polymer membranes include polyvinylidene fluoride. Examples of the structure of ultrafiltration membranes include flat membranes, spiral membranes, and hollow fiber membranes.

As such an ultrafiltration membrane, for example, commercially available products such as Amicon Ultra-0.5 ultracel (regenerated cellulose, manufactured by MILLIPORE) and 10K Microza SIP-0013 (polysulfone membrane, manufactured by Asahi Kasei Chemicals) can be used. Ultrafiltration can be performed by known methods. Examples of such known methods include centrifugal filtration and cross-flow filtration.

The "dialysis" according to the present invention can be performed using a dialysis membrane having a pore size that does not allow the passage of nucleotide-peptide complexes, but allows the passage of inorganic salts, sugars, and other low molecular weight molecules. Those skilled in the art can select the pore size of the dialysis membrane appropriately, taking into consideration the molecular weight of the nucleotide or peptide to be treated, but the pore size is preferably a molecular weight cutoff of 5 to 150 kDa, more preferably a molecular weight cutoff of 10 to 100 kDa, even more preferably a molecular weight cutoff of 20 to 70 kDa, and particularly preferably a molecular weight cutoff of 50 kDa.

The dialysis membrane may be, for example, a polymer membrane and structure made of the same material (such as cellulose-based) as the ultrafiltration membrane. In addition, as such a dialysis membrane, for example, commercially available products such as MF-Millipore Membrane (manufactured by Merck Millipore, catalog number: VSWP02500, pore size: 0.025 μm, material: cellulose mixed ester) and Spectra/Por7. Dialysis Membrane Pre-treated RC Tubing (manufactured by Funakoshi Co., Ltd., catalog number: 132112, pore size: 3.5 kDa, material: regenerated cellulose) can be used. In addition, dialysis can be performed by a known method. As such a known method, for example, a method in which a dialysis tube or dialysis cassette equipped with the dialysis membrane is immersed in a buffer exchange solution as an external solution can be mentioned.

The "chromatography" according to the present invention may be any method capable of separating nucleotide-peptide complexes from small molecules such as inorganic salts and sugars, and examples thereof include size exclusion chromatography (gel filtration chromatography, gel permeation chromatography, etc.), adsorption chromatography (affinity chromatography, etc.), hydrophobic interaction chromatography, hydrophilic interaction chromatography, and ion exchange chromatography. Furthermore, a person skilled in the art can appropriately select and prepare a chromatography capable of separating nucleotide-peptide complexes from small molecules such as inorganic salts and sugars based on differences in size, mass, adsorptive power, hydrophobicity, hydrophilicity, charge, etc., as well as the stationary phase and mobile phase that constitute the chromatography.

In the present invention, the buffer exchange may be performed in multiple stages, from the viewpoint that the activity of the nucleotide-peptide complex is more easily maintained and the microparticles described below are more easily supported stably by removing the inorganic salt and the sugar more slowly from the nucleotide-peptide complex solution. The number of times may be any number that ultimately results in a state in which the inorganic salt and sugar are substantially not contained, and may be, for example, two, three, four, or five times. In addition, the method used in the multiple buffer exchanges may be the same or different.

The "buffer exchange solution" is not particularly limited, and can be prepared by a person skilled in the art as appropriate. Usually, a buffer solution not containing inorganic salts and sugars (hereinafter also referred to as "buffer only") is used. In addition, when a buffer solution containing both inorganic salts and sugars is subjected to multi-stage buffer exchange, for example, first, the inorganic salts alone may be removed by buffer exchange with a buffer containing only sugars, and then the sugars may be removed by buffer exchange with only a buffer solution. In addition, first, the sugars alone may be removed by buffer exchange with a buffer containing only inorganic salts, and then the inorganic salts may be removed by buffer exchange with only a buffer solution. Furthermore, the inorganic salts and sugars may be finally removed by buffer exchange with buffer solutions having stepwise reduced concentrations of inorganic salts and/or sugars (more specifically, when a buffer solution containing 200 mM inorganic salts and 2% sugars is subjected to multi-stage buffer exchange, first, the buffer solution may be exchanged with a buffer containing 100 mM inorganic salts and 1% sugars, and then, the inorganic salts and sugars may be finally removed by buffer exchange with only a buffer solution).

Such multi-stage buffer exchange can also be performed continuously. For example, it can be performed by gradually reducing the concentration of inorganic salts and/or sugars in the buffer exchange solution using a continuous dialysis device. In addition, in affinity chromatography, multi-stage buffer exchange can also be performed continuously by passing a buffer exchange solution (so-called gradient buffer) in which the concentration of inorganic salts and/or sugars is gradually reduced through the stationary phase to which the nucleotide-peptide complex is bound.

Preparation of Microparticles Coated with Nucleotide-Peptide Complexes
The nucleotide-peptide complex solution from which inorganic salts and sugars have been removed is mixed with microparticles used in particle bombardment, so that the microparticles are covered with the complexes.

The material of the microparticles is not particularly limited, and examples include microparticles (metal microparticles) made of metals such as gold, tungsten, and magnetic particles (iron tetroxide, etc.). Among these, gold particles are preferred. The particle size of the microparticles is not particularly limited, but is, for example, 0.05 to 5 μm, preferably 0.1 to 3 μm, more preferably 0.2 to 1.5 μm, and even more preferably 0.4 to 0.8 μm.

In the present invention, "coated" refers not only to a state in which the entire surface of a microparticle is covered with the substance, but also to a state in which only a portion of the surface is covered (i.e., the substance is attached to a portion of the surface of the microparticle). Here, "a portion" refers to 50% or more of the surface of the microparticle, preferably 85% or more, more preferably 85% or more, and even more preferably almost the entire surface (e.g., 95% or more). Furthermore, "microparticles coated with a substance" may be in the form of a mixture of the substance and the microparticles.

(Coating of fine particles onto carrier)
The microparticles coated with the nucleotide-peptide complex are coated on a carrier for carrying a mixture containing the microparticles in a particle bombardment method.

The carrier is usually a substance also called a macrocarrier in the particle bombardment method. The material of the carrier is not particularly limited as long as it can support the fine particles and can be thin enough to release the fine particles when pressure is applied in the particle bombardment method. For example, materials used in so-called industrial films such as polyimide, polyethylene, polypropylene, polystyrene, polyethylene terephthalate, polytetrafluoroethylene, polyphenylene sulfide, etc. are included. The shape and size of the carrier are not particularly limited as long as it can be attached to the particle bombardment device described below, but it is usually a disk (disk) with a diameter of 2.5 cm. The thickness can be changed appropriately to the extent that the fine particles can be released depending on the type of material, but it is usually 5 to 100 μm, preferably 25 to 75 μm. Suitable carriers according to the present invention include commercially available products such as PDS-1000/He macrocarriers (manufactured by BioRad, diameter 2.5 cm, thickness 50 μm, material: Capron (registered trademark) (polyimide) manufactured by Toray DuPont Co., Ltd.). Coating of the carrier can usually be performed uniformly using a micropipette or the like.

(Degassing and drying)
The support of the microparticles coated with the nucleotide-peptide complex on the support is carried out by degassing and drying, which, as shown in the Examples below, overcomes the reduction in activity of the nucleotide-peptide complex and the instability of the support that occurs when drying by air drying or lyophilization.

Degassing and drying can be performed, for example, by reducing the pressure inside the system containing the carrier, as shown in the examples below. As processing conditions for such reduced pressure degassing, the pressure inside the system is preferably 500 hPa or less, more preferably 300 hPa or less, and even more preferably 100 hPa or less. The temperature inside the system is preferably 10 to 40°C, more preferably 15 to 30°C, and even more preferably room temperature (22 to 28°C). The processing time is preferably 5 to 60 minutes, and even more preferably 10 to 30 minutes. Degassing under such conditions can be performed using a commercially available degassing device (for example, a continuous vacuum degassing device).

<Kit of the Present Invention>
The present invention also provides a kit for use in the above-mentioned production method, which includes at least one of the following substances:
(A) the peptide and the nucleotide; (B) a neutral buffer solution containing an inorganic salt and/or a sugar; (C) an ultrafiltration membrane, a dialysis membrane, or a fixed phase for chromatography, used for the buffer exchange; and (D) a buffer exchange solution (a solution used for performing a buffer exchange with the lysate obtained in the step (1)).
(E) the fine particles; (F) a carrier onto which the mixed liquid is applied.

The kit of the present invention preferably includes, for example, (A), (C), (D), and (E), and more preferably includes (B) to (E), and particularly preferably includes all of (A) to (F). Furthermore, the kit of the present invention may include instructions showing the above-mentioned manufacturing method, etc.

<Carrier of the present invention>
As described above, the manufacturing method of the present invention can produce a carrier in which a substance (nucleotide-peptide complex) to be introduced into cells, which is used in the particle bombardment method, covers the microparticles while maintaining its activity, and the microparticles are stably supported. Therefore, the present invention can also provide a carrier manufactured by the above method.

In other words, the present invention provides a carrier carrying fine particles coated with a substance used in particle bombardment, as shown in the examples below, in which the substance is a complex of peptide and nucleotide that is introduced into cells by particle bombardment, and in which the surface carrying the fine particles has precipitates of less than 100 μm in a region within 10 mm from the center.

The "precipitate" is a substance composed of inorganic salts and/or sugars, which is precipitated on the surface of the carrier on which the microparticles are supported, and may be a mixture of a buffer, the introduced substance, and the microparticles. The size is less than 100 μm, preferably 20 μm or less, and more preferably 5 μm or less. The size of the precipitate refers to the longest diameter of the substance. The precipitates are preferably 10 or less, more preferably 5 or less, even more preferably 3 or less, and particularly preferably 0, in an area within 10 mm from the center of the carrier. The "center" refers to the geometric center.

In addition, in the layer of the buffer and the fine particles coated with a substance that is provided on the surface of the carrier of the present invention, the weight ratio of the buffer to the fine particles is usually buffer:fine particles=1:1 to 1:10, and preferably 1:2 to 1:5.

<Particle bombardment method of the present invention>
As described above, according to the present invention, salts and the like are not precipitated on the surface of the carrier, and the activity of the nucleotide-peptide complex can be maintained, making it possible to introduce the complex into cells and allow it to function while minimizing damage caused by the precipitates.

Therefore, the present invention also provides a method for introducing a nucleotide-peptide complex into a cell by particle bombardment, which includes a step of applying pressure to the carrier of the present invention, thereby bombarding the microparticles released from the carrier into the cell.

Below, an example of an embodiment of the particle bombardment method of the present invention will be described with reference to Figures 1A and 1B.

In the particle bombardment method, for example, before the nucleotide-peptide complex is shot, the inside of the particle bombardment device 1 is made negative, and gas is supplied to the gas acceleration tube 2 until the rupture disk 3 reaches the rupture pressure. Next, as shown in FIG. 1B, the gas shock pressure generated by the rupture disk 3 bursting (rupture disks 3a and 3b after bursting) causes the carrier 4 carrying the microparticles 5 coated with the above-mentioned nucleotide-peptide complex on its underside to move toward the target (cell 7). Then, the carrier 4 stops at the stopping screen 6, and only the carried microparticles 5 are shot into the target (cell 7) placed on the support 8.

The present invention is not limited to the particle bombardment device of the form shown in the figure, and as long as the device can eject fine particles supported on the carrier at high speed, regardless of its form or ejection method, a person skilled in the art can set the appropriate conditions to suit the device and practice the present invention.

In the present invention, the "cells" into which the nucleotide-peptide complex is introduced are not particularly limited and may be of animal or plant origin.

There are no particular limitations on the "plant" from which the cells are derived, and examples include angiosperms, including dicotyledonous and monocotyledonous plants, gymnosperms, herbaceous plants, and woody plants. More specific examples of plants include tomato, tobacco, bell pepper, chili pepper, eggplant, and other nightshades; gourds, such as cucumber, pumpkin, and watermelon; lettuce, broccoli, Chinese cabbage, and Arabidopsis; celery, parsley, lettuce, and other fresh and spicy vegetables; onions, onions, garlic, and other alliums; beans, such as soybeans, peanuts, kidney beans, peas, adzuki beans, mung beans, cowpeas, and broad beans; strawberries, melons, and other fruit vegetables; taproots, such as radishes, turnips, carrots, and burdock; tubers, such as taro, cassava, potato, sweet potato, and Chinese yam; rice, corn, wheat, sorghum, barley, and rye. cereals such as wheatgrass, wheatgrass, and buckwheat; soft lettuce such as asparagus, spinach, and mitsuba; petals such as lilies, lisianthus, stock, carnations, and chrysanthemums; turfgrass such as bentgrass and zoysiagrass; oil crops such as rapeseed, peanut, rapeseed, and jasmine; fiber crops such as cotton and rush; forage crops such as clover, dent corn, and alfalfa; deciduous fruit trees such as apples, pears, grapes, peaches, and kiwifruit; citrus fruits such as satsuma mandarins, oranges, lemons, and grapefruit; and woody plants such as azalea, azalea, cedar, poplar, rubber tree, ginkgo, and pine.

Such cells derived from plants include plant cells present in any tissue or plant cells derived from any tissue. Examples of such tissues include leaves, roots, root tips, anthers, flowers, seeds, pods, stems, shoot tips, embryos, and pollen. Furthermore, the method of the present invention can also target artificially treated plant cells (e.g., cultured cells, callus, and suspension culture cells). In addition, in the present invention, "pollen" includes not only mature pollen but also immature pollen. Furthermore, cells that constitute pollen (agonist cells, sperm cells, pollen tube cells (vegetative cells), etc.) are also included in the pollen that is the target of the substance introduction of the present invention.

The present invention will be described in more detail below with reference to examples and reference examples, but the present invention is not limited to the following examples.

Reference Example 1 Preparation of freeze-dried protein and its evaluation In order to introduce RNA protein complexes (hereinafter also referred to as "RNP") into pollen using the particle bombardment method, RNP must be mixed with gold particles and dried. Therefore, first, Cas9 protein was used as a protein constituting RNP, and the solution composition for freeze-drying the protein was examined by the following method.

(Buffer replacement and freeze-drying of protein solutions)
The composition of the commercially available Cas9 protein (Fasmac Co., Ltd.) with a concentration of 5 μg/μl was dialyzed to obtain the desired solution composition. Specifically, the Cas9 protein was filled into a dialysis membrane (Fujifilm Wako Pure Chemical Industries, Ltd.) with a molecular weight cutoff of 3.5 KDa, and the solution was dialyzed as a dialysis buffer to replace the buffer. The Cas9 protein solution replaced with the desired solution composition was frozen at -80°C and then freeze-dried. The freeze-drying method was performed using a freeze-drier (model number: PFR-1000, manufactured by EYELA Tokyo Rikakikai Co., Ltd.) in accordance with its usage instructions. The commercially available product was also used as a positive control. The pH was all set to 7.5.

(Cleavage activity evaluation)
In order to evaluate the activity of the freeze-dried Cas9 protein of each composition, ultrapure water was added to the freeze-dried Cas9 to return it to 5 μg/μl Cas9 protein, and 3,500 ng of the protein was used to confirm the cleavage activity in order to evaluate the activity of the protein.

As a reaction system for evaluating the cleavage activity, 3500 ng of the Cas9 protein was added to a solution consisting of 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10 mM MgCl ₂ and 1 mM DTT, 300 ng of double-stranded DNA containing the target sequence of genome editing (DNA consisting of the sequence described in SEQ ID NO: 1) as a template, 280 ng of crRNA effective for the target sequence (RNA consisting of the sequence described in SEQ ID NO: 2), and 467 ng of tracrRNA, and prepared. In addition, a solution in which only 300 ng of the template DNA was added to the solution instead of the genome editing system was used as a negative control.

The reaction system was then left to stand at 37°C for 1 hour, after which 1/10 of the reaction volume of STOP solution (30% glycerol, 1.2% SDS, 250 mM EDTA) was added and the reaction was stopped by reacting at 37°C for 15 minutes.

After further incubation at 60°C for 5 minutes and then on ice for 5 minutes, 10 μl of each reaction volume was loaded onto a 5-20% SDS-PAGE gel and electrophoresed in Tris-acetate EDTA buffer (40 mM Tris-acetate, 1 mM EDTA). The gel after electrophoresis was stained with SYBR safe (Thermo Fisher Scientific) to detect DNA in the gel. The amount of template DNA detected in each reaction system was converted into a relative value when the amount of template DNA in the negative control was set to 100, and the obtained value was taken as the cleavage rate of the template DNA.

In addition, instead of the Tris-acetate EDTA buffer, SDS buffer (25 mM Tris, 0.1% SDS, 191 mM Glycine) was used, and the gel was subjected to electrophoresis, after which the amount of Cas9 protein in the gel was detected by CBB staining.

As a result, although not shown in the figure, it was revealed that when the Cas9 protein was freeze-dried in a solution consisting of 20 mM Tris-HCl, 200 mM KCl, and 2% sucrose, the cleavage activity was maintained even after drying (the cleavage rate of the positive control (commercial product) was 86.1%, while that of the dried Cas9 protein was 89.4%).

Similar results were obtained when 20 mM HEPES was used instead of the 20 mM Tris-HCl. Furthermore, no significant change in the amount of Cas9 protein was observed before and after the freeze-drying.

Reference Example 2: Study on the stability of RNP when salt and sugar are removed In Reference Example 1, an example of a solution for maintaining protein stability during lyophilization was shown, but the salt concentration in the solution is high. Therefore, a large amount of salt will precipitate in the particle bombardment method, which may cause problems when introducing the RNP (see Reference Example 4 below).

Therefore, conditions for removing salt while maintaining protein stability were examined. Specifically, 30 μl of the solution (20 mM Tris-HCl, 200 mM KCl, 2% sucrose) containing 3000 ng of Cas9 protein was prepared, and salt and sugar were removed by membrane dialysis under the following different conditions. In addition, the solution containing not only 3000 ng of Cas9 protein but also crRNA (240 ng) and tracrRNA (400 ng) (these RNAs are also referred to as "RNAmix") was prepared, and salt and sugar were removed by membrane dialysis under the following conditions while keeping the RNP in a complex state.
(conditions)
1: A solution containing 3000 ng of Cas9 was replaced with 20 mM Tris-HCl by membrane dialysis.
2: A solution containing 3000 ng of Cas9 was replaced with pure water (H ₂ O) by membrane dialysis.
3: A solution containing 3000 ng of Cas9 protein and RNAmix was replaced with 20 mM Tris-HCl by membrane dialysis.
4: A solution containing 3000 ng of Cas9 protein and RNAmix was replaced with pure water (H ₂ O) by membrane dialysis.
5: A solution containing 3000 ng of Cas9 protein and RNAmix was replaced with a solution consisting of 20 mM Tris-HCl and 2% sucrose by membrane dialysis.
6: The solution containing 3000 ng of Cas9 protein and RNAmix was replaced with a solution consisting of 2 mM Tris-HCl and 0.2% sucrose.

The membrane used was an MF-Membrane Filter 0.025 μm, VSWP (Merck Millipore). The container was filled with the buffer to be replaced, the membrane was floated on the water surface, and the Cas9 protein solution was placed on the membrane. After buffer replacement, crRNA, tracrRNA, and template DNA were mixed under conditions No. 1 and 2, and only template DNA was added under conditions No. 3-6, and cleavage activity was evaluated in the same manner as in Reference Example 1. The results obtained are shown in Figure 2.

As a result, as shown in Figure 2, removal of salt and sugar by Cas9 protein alone significantly reduced the cleavage activity. On the other hand, with RNP, the salt and sugar concentrations could be reduced and removed under conditions No. 3, 5, and 6 without any reduction in activity. However, when replaced with _H2O , even RNP could not retain activity, resulting in a reduction in cleavage activity.

(Reference Example 3) Investigation of the buffer replacement method and drying method when removing salts and sugars The results shown in Reference Example 2 suggest that it is possible to dry RNP while retaining its activity using only 20 mM Tris-HCl, which also removes sugars. Therefore, we investigated the buffer replacement method and the drying method as a method for removing salts and sugars more stably.

Specifically, 30 μl of the above solution (20 mM Tris-HCl, 200 mM KCl, 2% sucrose) containing 3500 ng of Cas9 protein, 280 ng of crRNA, and 467 ng of tracrRNA was prepared, and salts and sugars were removed by ultrafiltration under the conditions shown below while keeping the RNP in a complex state.
1: The medium was replaced with a solution of 20 mM Tris and 2% sucrose by ultrafiltration, then replaced with 20 mM Tris, and then freeze-dried.
2: The medium was replaced with a solution consisting of 20 mM Tris and 2% sucrose by ultrafiltration, then replaced with 20 mM Tris, and then air-dried.
3: Template DNA only (negative control)
4: Cas9 protein 3500 ng only In the ultrafiltration method, an Amicon Ultra-0.5 ultracel, 50 kDa (Merck Millipore) column was used. In addition, to examine the drying method, lyophilization (similar to Reference Example 2) or air drying (Air-Dry) was performed as described above. After drying, ultrapure water was added to the dried product, and template DNA was added to evaluate the cleavage activity, as in Reference Example 1. The results obtained are shown in FIG. 3.

As a result, as shown in Figure 3, extremely high cleavage activity was observed under condition 1, i.e., when the buffer solution was exchanged by ultrafiltration and then freeze-dried. On the other hand, as shown by the cleavage activity under condition 2, the cleavage activity was reduced by half when the sample was air-dried even after the buffer solution exchange. Therefore, it was also made clear that freeze-drying is more preferable than air-drying as a drying method after buffer solution exchange.

Next, we used condition 1 to create an RNP-loaded disk for the particle bombardment method.

Specifically, 3500 ng of Cas9, 467 ng of tracrRNA, and 280 ng of crRNA were mixed in 30 μl of the above solution (20 mM Tris-HCl, 200 mM KCl, 2% sucrose) to prepare RNP. The solution was then replaced with a solution consisting of 20 mM Tris and 2% sucrose by ultrafiltration, and then replaced with 20 mM Tris. Meanwhile, 0.6 μm diameter gold particles (manufactured by BIO-RAD) were washed with 100% ethanol and suspended in sterile water to prepare a 30 mg/ml gold solution. Then, 10 μl of gold solution was added per shot to 20 μl of the RNP solution after the buffer exchange, and 30 μl was loaded onto one particle bombardment macrocarrier (BioRad PDS-1000/He macrocarrier) and frozen at -80°C. The disk was then freeze-dried to prepare a disk.

As a result, as shown in Figure 4, after loading and drying the RNP prepared in this manner, a white precipitate was observed in the center of the disk surface (the area indicated by the triangle in the figure), making it clear that the disk was prone to crumbling.

(Example 1) Study on drying method 2
As described above, the freeze-drying method can be said to be a desirable drying method from the viewpoint of maintaining the activity of RNP, but it has become clear that the freeze-drying method is unstable when applied to the particle bombardment method.

Therefore, we attempted to dry the sample in a state between air drying and freeze drying using the degassing drying method. Specifically, we prepared RNP-loaded disks using the degassing drying method instead of the freeze drying method used in Reference Example 3. In this degassing drying method, a mixed solution of RNP and gold particles was loaded into a macrocarrier, and the solution was dried at 25°C for approximately 20 minutes using a freeze dryer under a reduced pressure of approximately 100 hPa (approximately 1/10 of 1 atmosphere) with the RNP and gold particles on the carrier remaining in solution. Under these conditions, the sample gradually dries from the liquid without freezing during drying.

On the other hand, for comparison, as shown in Reference Example 3, in the freeze-drying method, the sample was frozen and then sucked under as much vacuum as possible to dry the sample in a frozen state, and an RNP-loaded disk was prepared.

As a result, as mentioned above, in the disks prepared by freeze-drying, a powder-like substance that appeared to be a precipitate of the solution appeared in the center (pointed out by a triangle in the figure), and the surrounding area was also brittle (see Figure 4). On the other hand, in the disks prepared by degassing and drying, as shown in Figure 5, no precipitation was observed as in the freeze-drying method, and the sample (mixture of RNP and gold particles, etc.) was uniformly loaded and dried on the macrocarrier, was less brittle than the one shown in Figure 4, and the sample was firmly supported.

30 μl of the above solution (20 mM Tris-HCl, 200 mM KCl, 2% sucrose) containing 3500 ng of Cas9 protein, 280 ng of crRNA, and 467 ng of tracrRNA was prepared, and the RNP was in a complex state, salts and sugars were removed by buffer exchange, and then freeze-dried or degassed and dried under the following conditions. After drying, ultrapure water was added to the dried product, and template DNA was added, as in Reference Example 1, to evaluate the cleavage activity.

As a result, as shown in Figure 6, the RNP cleavage activity remained high even when the drying method was changed from freeze-drying to degassing.

Reference Example 4: Consideration of solution composition In the above Example 1, it became clear that the degassing and drying method was effective from the viewpoint of maintaining the stability of the RNP-supported disk in the particle bombardment method.

Next, as shown below, the composition of the RNP solution was changed to prepare RNP-supported disks, which were then observed under a fluorescent microscope.
1. Conditions shown in Example 1 Cas9 3500ng, tracrRNA 467ng and crRNA 280ng were mixed in 30μl of the above solution (20mM Tris-HCl, 200mM KCl, 2% sucrose) to prepare RNP. Next, the buffer was exchanged with a solution consisting of 20mM Tris and 2% sucrose by ultrafiltration, and then with 20mM Tris. Meanwhile, 0.6μm diameter gold particles (manufactured by BIO-RAD) were washed with 100% ethanol and suspended in sterile water to prepare a 30mg/ml gold solution. Then, 10 μl of gold solution was added per shot to 20 μl of the RNP solution after the buffer exchange, and 30 μl of the solution was loaded onto one macrocarrier for particle bombardment, followed by the degassing and drying method to prepare an RNP-supported disk.
2. Conditions equivalent to those described in Non-Patent Document 3 An RNP-supported disk was prepared under the conditions shown in Example 1 above, except that a solution consisting of 20 mM HEPES (pH 7.5) and 150 mM KCl was used instead of the above solution, and the RNP solution was prepared without performing the two-stage exchange by ultrafiltration.

As shown in Figure 7, the sample loading area in Figure 5 was observed with an optical microscope, and it was revealed that under the conditions shown in Example 1, the loaded mixture was dried by degassing and drying, and was supported in a very fine, dense, and uniform state. In other words, in the black area of approximately one-quarter of a circle surrounded by the red line in the figure (below the color display), black fine particles were uniformly supported, and it was observed that the gold particles coated with the substance were present all over the surface without forming lumps.

On the other hand, in the disks prepared under conditions equivalent to those described in Non-Patent Document 3, as shown in Figure 8, there were many precipitates of 100 μm or more (pointed to by triangles in the figure), possibly due to the influence of salt, and there were also large precipitates. Note that at the edge of the circle shown in Figure 8, the gold particles coated with the substance were gathered together and were incorporated into the precipitate, forming black precipitates (area (b) in the figure). Since cells are usually 100 μm or less in size, it is estimated that with such carriers, not only would the precipitates make it difficult to introduce RNP into the cells, but the impact of these precipitates would cause significant damage to the cells.

From the above, it became clear that in order to minimize the salt and sugar content, which have a significant effect on precipitation, it is necessary to remove salts, etc. by buffer exchange such as ultrafiltration. Furthermore, by using the degassing drying method as a subsequent drying method, the precipitation can be suppressed, making it possible to prepare a cassette into which the nucleotide-peptide complex can be introduced with minimal damage to the cells. In addition, since the fragility of the surface could be eliminated, it became possible to prepare a cassette that can be transported and stored.

As explained above, according to the present invention, it is possible to provide cells with new functions by introducing a substance (nucleotide-peptide complex) into the cells. Therefore, it is also very useful in the production and development of biomass, functional food ingredients, pharmaceutical materials, etc., and the present invention will make a significant contribution to various industrial applications.

1...particle bombardment device, 2...gas acceleration tube, 3...rupture disk, 3a...rupture disk after rupture, 3b...rupture disk after rupture, 4...carrier, 5...microparticles coated with substance, 6...stopping screen, 7...cells, 8...support.

Claims (4)

A method for producing a carrier carrying fine particles coated with a substance, which is used in a particle bombardment method, comprising the steps of:
the substance is a peptide-nucleotide complex that is introduced into cells by particle bombardment;
(1) dissolving the peptide and the nucleotide in a neutral buffer containing an inorganic salt and/or a sugar;
(2) removing the inorganic salts and the sugars from the lysate obtained in step (1) by buffer exchange;
(3) mixing the solution obtained in step (2) with the microparticles;
(4) applying the mixture obtained in step (3) onto the surface of a carrier ; and
(5) A step of drying the carrier coated with the mixed solution in the step (4) by a degassing and drying method ,
the inorganic salt is a sodium salt, a potassium salt or an ammonium salt,
the sugar is sucrose, maltose or trehalose;
The method, wherein the peptide is a Cas9 protein and the nucleotide is a guide RNA . 2. The method of claim 1 , wherein the neutral buffer is a Tris buffer of pH 7-8 or a HEPES buffer of pH 7-8. A method for introducing a substance into a cell by particle bombardment, comprising:
A step of producing a carrier carrying fine particles coated with the substance by the method according to claim 1 or 2; and
The method includes a step of applying pressure to the carrier produced in the step , thereby shooting the microparticles released from the carrier into the cells. A kit for use in the method according to claim 1 or 2 , comprising at least one substance selected from the group consisting of substances (A) to (F) below: (A) the peptide and the nucleotide; (B) a neutral buffer solution containing an inorganic salt and/or a sugar; (C) an ultrafiltration membrane, a dialysis membrane or a chromatography immobilized phase used for the buffer exchange; (D) a solution used for performing the buffer exchange with the lysate obtained in the step (1); (E) the fine particles; and (F) a carrier on which the mixed solution is applied.
(wherein the inorganic salt is a sodium salt, a potassium salt or an ammonium salt, the sugar is sucrose, maltose or trehalose, the peptide is a Cas9 protein, and the nucleotide is a guide RNA) .