RU2663347C1 - Method of delivering biologically active macromolecules into the cells of plants - Google Patents

Abstract

FIELD: biochemistry.SUBSTANCE: invention relates to the field of biochemistry, in particular to a method for delivering macromolecules selected from nucleic acids, proteins or nucleoprotein complexes to a plant cell. Method comprises providing a plant cell having a cell wall, coating a surface of a 600–800 nm chitosan nanoparticle with a nucleic acid, a protein or a nucleoprotein complex, bringing the plant cell having the cell wall and said coated nanoparticle into contact with each other and allowing the said nanoparticle to be absorbed into the plant cell containing the cell wall.EFFECT: invention allows efficient delivery of macromolecules selected from nucleic acids, proteins or nucleoprotein complexes to a plant cell having a cell wall, with the possibility of subsequent reprogramming of plant cells and/or editing their genome.17 cl, 9 dwg, 8 ex

Description

The scope of the invention

The invention relates to the field of biotechnology, physico-chemical biology, molecular biology, and is aimed at developing an effective method for the delivery of biologically active macromolecules (proteins, nucleic acids of various sizes and nucleoprotein complexes of proteins with nucleic acids) in plant cells. In accordance with the present invention, nanoplatforms based on chitosan particles infiltrated into plant cells have been obtained, functionalized with biologically active macromolecules, which can be used in various bio-nanotechnologies, for example, for their subsequent use in editing the plant genome by a method that excludes uncontrolled introduction of foreign genetic material, protect agricultural plants from various pathogens, including bacteria, oomycetes, fungi and viruses, as well as for eticheskoy plant transformation.

Description of the invention

Terminology: All terms and definitions given in the text should be understood in the sense in which they are usually applied in the relevant fields of science and technology.

Certain definitions, specifically mentioned, are not exceptions and are provided only for the purpose of facilitating understanding of the essence of the invention.

Biomolecule Delivery Efficiency — Percentage of target biomolecules entering the plant cell, determined by introducing fluorescent / fluorescently labeled molecules.

The effectiveness of genomic editing is the percentage of loci in the genome of plant cells into which directed mutations have been introduced.

Biologically active macromolecules, biomolecules or biopolymers are polymers that occur naturally in nature and are part of living organisms.

The ever-growing world population and the declining fund of agricultural arable land stimulate research aimed at improving the efficiency of agriculture. Standard means of increasing the yield of agricultural and horticultural crops include breeding techniques based on the identification of plants characterized by the desired traits. Molecular biology technologies based on the manipulation of genetic material (nucleic acids in the form of DNA or RNA) and the subsequent introduction of this genetic material into a plant allow the creation of cultures characterized by various improved economic, agrotechnical and horticultural characteristics.

The objective of the present invention was to develop a technically simple universal method for the delivery of biologically active macromolecules to plant cells, which is applicable for the delivery of a variety of macromolecules, including short RNA, DNA, proteins and protein complexes with nucleic acids, with the efficiency that allows you to regulate various cellular processes, including including the activity of cell genes and the protective response of plants to infection by various pathogens.

As a promising tool for the delivery of nucleic acids, proteins and nucleoproteins into the cell, polymer systems, including nanoparticles and nanocapsules, are currently considered [Markvicheva EA Chitosan and its derivatives in bioencapsulation. Chitin and chitosan: production, properties and application. M .: Publishing House of Science, 2002.p. 315-326.]. Documents WO / 2010/118077, WO / 2009/046384, WO / 2012/006439, WO / 2012/006443, WO 2008/083233, WO 2011US57813A disclose the use of nanoparticles for DNA delivery and plant transformation. Also known is a mediated method of delivering sequence-specific nucleases to plant cells mediated by gold nanoparticles, disclosed in documents RU 2612156 C2 and RU 2556376 C2 (DAU AGROSAIENISIS, (US)). Unlike the known method, in the present invention, chitosan is proposed as a material for creating nanoparticles for use in the delivery of macromolecules to plant cells. Chitosan is widely used in agriculture in the development of plant protection products, however, to date, no methods have been developed for its use for the efficient delivery of macromolecules to plant cells. Chitosan, in which glucosamine residues predominate, is a natural polymer with a wide range of biological properties, including biocompatibility and biodegradability. Recently, chitosan is considered as one of the promising materials in the creation of polymer matrices for delivery in drugs and DNA to animal cells.

The systems of delivery to animal cells with DNA vectors embedded in them have been developed on its basis and have several advantages: ligands that provide highly specific interaction with cellular receptors can be covalently attached to their surface; you can enter into the cell not one but several DNA plasmids in one nanoparticle or nanocapsule; nucleic acid can be protected from enzyme digestion; DNA plasmids acquire a compact form, which prevents their mechanical rupture and facilitates penetration through cell membranes; such nanoparticles can be freeze-dried and stored for a long time in this form without loss of activity [Gene Ther. 2004; v. 11: 19: 1441-1452.]. The methods for introducing genetic material, DNA, RNA, proteins, and nucleoprotein complexes into plant cells are significantly different from those used for animal cells. The main obstacle to macromolecules is the cell wall. Its chemical composition and features of the arrangement of its constituent components contribute to the binding of nucleic acids and proteins of various structures, thereby limiting their access to the cell membrane. When using non-viral vectors, for example, carrying the target gene of bacterial plasmid DNA, the efficiency of direct transformation is very low. The experiments conducted in the 1970s and 1980s on introducing suspensions of DNA molecules into the ovary of flowers or young flower shoots using a syringe (macroinjection), soaking dry plant seeds or pollen in such solutions, and the use of electrophoresis to enhance the penetration of DNA into seedling tissues showed that the frequency of formation of transformants is extremely low (less than 2%). Alternative are methods for introducing DNA into protoplastic (devoid of a cell wall) plant cells, where a complex of enzymes of fungal or bacterial origin, consisting of pectinliases and cellulases, is used to remove the cell wall of plant cells.

Currently, they often use the physical method of delivering DNA to a cell - bombarding it with microparticles (bio-ballistics or abbreviated biolistics). DNA on particles with a diameter of 0.4 to 1.2 μm is fixed by treatment with solutions of nucleic acid and CaCl ₂ , or spermidine, or polyethylene glycol. Particles coated with DNA are accelerated to a speed of 300-600 m / s using special instruments and the flow is directed to isolated cells, fragments, or the whole plant. To create a flow of flying particles, gases generated by the combustion of gunpowder, compressed air or helium are used. Particles pierce the cell wall and membranes of the plant cell. The DNA delivered to the cells partially goes into a free state and interacts with the DNA molecules in the nucleus, mitochondria, or plastids.

The microparticle bombardment method allows you to transform plants of almost any species. But it’s known that it occurs at a low frequency, and the integration and expression of foreign genes may depend on the vector used for their introduction. For example, the frequency of transformation is increased if linear rather than circular DNA is used. In addition, when microparticle bombardment, high molecular weight plasmids (more than 10 kb) can fragment, so the level of expression of foreign genes will be lower than in the case of smaller plasmids.

As microparticles for the bombardment of plant tissues, use spherical particles of noble metals - gold or tungsten, since they have the necessary mass and hardness and are not toxic.

In the present invention, chitosan is used as a material for creating carrier particles of macromolecules, which, due to its polycationic nature, is able to form polyelectrolyte complexes with negatively charged polymers, such as nucleic acids, proteins, nucleoproteins (Applied Biochemistry and Microbiology 2005; v. 41: 1: 9-16. Biomacromolecules 2004; v. 5: 928-936.). The inventors have developed an original technique that allows the use of chitosan for the construction of nanoparticles-carriers of macromolecules (DNA, nucleoprotein complexes and proteins) and their delivery to the plant cell.

In particular, using the method of delivery of macromolecules disclosed in the present invention, the inventors propose editing the genome of higher plants, for which CRISPR-Cas9 system elements can be used. The ability of chitosan particles to bind macromolecules of various compositions and sizes with high efficiency is extremely important in this case: for the CRISPR system to work, a specially processed CRISPR-PHK (crPHK) is necessary. CRISPR - genome editing methods are based on the use of DNA interference, directing (guiding) RNA and targeting specific DNA sequences. A number of important discoveries regarding the design of type II CRISPR systems (in particular, the clarification of the need for Cas9 protein and additional, in addition to crRNA, small RNA called tracrPHK), made it possible to experimentally test the first artificially developed type II CRISPR system in 2012. Artificial CRISPR-Cas systems can work not only in bacterial and in vitro cells, but also in eukaryotic cells.

The expected technical result of the present invention was the development of a unified procedure for the delivery of macromolecules to plant cells, the expansion of the methodological arsenal of methods and means of delivery of macromolecules, in particular nucleic acids, proteins and nucleoprotein complexes, to the plant cell, with the possibility of subsequent reprogramming of plant cells and / or editing of their genome.

Description of drawings

FIG. 1. Phase-contrast image of chitosan particles obtained using a fluorescence microscope (A), fluorescence microscopy of chitosan particles functionalized with bovine serum albumin labeled with fluorescein isothiocyanate (FITC) (B) and fluorescence microscopy of chitosan particles functionalized with fluorescence drug Su-3 (B). Effective binding of chitosan particles to fluorescently-labeled protein and RNA molecules is seen.

FIG. 2. Infiltration of plants Nicotiana benthamiana FITZ-BSA immobilized on chitosan particles or gold particles (in the presence of chitosan particles).

FIG. 3. Infiltration of tRNA labeled with Cu3 fluorescent dye and immobilized on chitosan particles into Nicotiana benthamiana plant cells. The cells of the conducting beam are brightly colored.

FIG. 4. Evaluation of the effectiveness of the method for the delivery of plasmid DNA encoding ZFB into plant cells by infiltration with chitosan particles.

FIG. 5. Electrophoregram library before and after cleaning.

M is a molecular weight marker for DNA.

1 bАu - biolistic bombardment with gold nanoparticles coated with Cas9-sgRNA complexes

2 bН2O - biolistic bombardment with Cas9-sgRNA complexes without nanoparticles (- control)

3 iChit - vacuum infiltration with Cas9-sgRNA complexes on chitosan nanoparticles

4 iH2O - vacuum infiltration with Cas9-sgRNA complexes without nanoparticles (-control)

FIG. 6. Mapping of reeds to identify editing of the potato coilin gene.

FIG. 7. Morphogenic callus of potato, a variety of Chicago, separated from the stem explant for particle infiltration.

FIG. 8. Regeneration of shoots from morphogenic callus of potato, cultivar Chicago, after infiltration of a suspension containing particles of chitosan (0.25%), Cas9 and gRNA PDS.

FIG. 9. T7E1 DNA test of morphogenic callus of potato, which underwent editing of the phytoendesaturase gene by infiltration with chitosan nanoparticles, with the Cas9-sgRNA complex immobilized on their surface. PCR is an amplified fragment of the phytoendesaturase gene of morphogenic callus of potato that has been edited. T7E1 - it is also treated with T7 endonuclease 1. The presence of cutting the target DNA indicates the presence of genomic editing at the desired locus.

Disclosure of invention

The technical result is achieved due to the optimal combination of techniques and means used in the delivery procedure: in particular, to obtain optimal chitosan particles, their binding to macromolecules of various nature (nucleic acids, nucleoprotein complexes and proteins under optimal conditions, determined by the structure and properties of the delivered macromolecule , and introducing thus loaded nanoparticles into plant cells.

Obtaining particles of chitosan and their functionalization.

Chitosan (Sigma, CAS Number 9012-76-4) at a concentration of 1.75 mg / ml was dissolved in 30 mM Na-acetate buffer pH 4.5 (the buffer was prepared by adjusting the pH of a solution of 30 mM CH ₃ COONa to 4.5 10% solution of CH ₃ COOH) with constant shaking until complete dissolution of chitosan. Then, an aqueous solution of sodium tripolyphosphate (TPF, Galreakhim) was added to the solution at a concentration of 1 mg / ml in a ratio of chitosan: TPF equal to 5: 2. These conditions led to the formation of a homogeneous preparation of chitosan particles with a diameter of 600-800 nm (phase-contrast image obtained using an Axiovert 200M motorized inverted fluorescence microscope (Carl Zeiss, Germany), Fig. 1a. For some methods (indicated below), the initial solution particles of chitosan with a pH of 4.5 was neutralized by adding 10% NaOH solution to a pH of 6.5. To the resulting solution of chitosan particles were added solutions of protein, RNA or DNA (concentrations are indicated in the corresponding examples), mixed and voiced (processed ultrasonically with a frequency of 50/60 Hz) at 25 ° C for 10 minutes using an ultrasonic washing Elmasonic s300h (Germany).

The loading efficiency of nanoplatforms was evaluated by fluorescence microscopy, based on the number of fluorescent particles relative to the total number of particles visible in the light field (Fig. 1). The signals of fluorescent dyes (FITC and Su-3), which were used to label proteins and RNA, respectively, using standard techniques, were recorded using an Axiovert 200M motorized inverted fluorescence microscope (Carl Zeiss, Germany) controlled by the Axiovision 4.5 software package (Carl Zeiss Microimaging, Germany ) and equipped with Plan-Neofluar lenses with magnifications of 10x and 20x. Signals were detected in the green and red channels. In parallel, a phase-contrast image of each field of view was recorded. For image registration, an ORCA II ERG-2 digital camera (Hamamatsu Photonics, Japan) was used. Additionally, fluorescently-labeled proteins and RNA in plant cells were detected using a confocal laser scanning microscope, model Leica TCS SP5 (Leica Mycrosystems, Heidelberg, Germany). The cells were detected green fluorescent protein (GFP). To excite the glow, an argon laser with a wavelength of 488 nm was used.

The evaluation showed that chitosan particles effectively bind fluorescently labeled protein / RNA under experimental conditions. Fluorescent dye-labeled proteins and RNA bound to chitosan particles are shown in FIG. 1b, c. The resulting nanoplatforms remained stable and functional in solution for 24-48 hours.

To implement the method, it is not fundamental how the chitosan particles are functionalized — for example, it can be provided by electrostatic adsorption, conjugation with a ligand on the surface of the nanoparticle, coating the nanoparticle with a small cofactor, which the delivered RNA, DNA, protein or nucleoprotein macromolecule can recognize and bind , and direct conjugation of a macromolecule with a nanoparticle.

Obtaining experimental plants. In the experiments, plants of Nicotiana benthamiana and potato Solanum tuberosum were used. Plants were grown from seeds / minicubers and kept in a greenhouse at 16 hours a day, with a minimum daily temperature of 28 ° C and a minimum temperature of 22 ° C at night.

Infiltration of functionalized chitosan particles into plant cells.

Infiltration was carried out with chitosan particles functionalized with protein / RNA / DNA / RNP or chitosan particles (0.5% solution) with the addition of a solution of gold particles ((BioRad, Microcarrier Gold 0.6 μm in size) functionalized with protein / RNA / DNA / RNP. The resulting suspensions / particle solutions were pre-voiced at 25 ° C for 10 minutes at a frequency of 50/60 Hz using an Elmasonic s300h ultrasonic cleaner (Germany). Mechanical and vacuum methods were used to infiltrate plant tissues. acroinjection using an injectable sterile syringe. Vacuum infiltration can be carried out by placing the tissue in the desiccator and creating a vacuum in it, after which, when depressurization and a sharp increase in pressure, the applied suspension is thrown into the tissue. The preparation of chitosan particles functionalized with protein / RNA / DNA or a protein-RNA complex, protein-DNA, was infiltrated into the lower surface of the sheet through slight damage to the external epidermis. The infiltration area was at least 1 cm in diameter. Vacuum infiltration was performed using a PDS-1000 / Not gun (BioRad). For plant samples, a pressure of 28-30 inches of mercury was used, in accordance with the manufacturer's instructions. Samples (5 × 5 mm leaf plate die cuts, apical meristems (about 10 meristems) or axillary kidneys) were placed in a PDS-1000 / He gun chamber, up to 100 μl of the prepared preparation was applied to the sample, the pressure in the chamber was raised to the recommended level, maintained for 60 seconds, and then reduced the pressure to normal levels. The plant cells / tissues infiltrated with functionalized chitosan particles were kept in a climatic chamber or at room temperature. Evaluation of the results was carried out 2-3 days after infiltration by fluorescence microscopy and laser confocal microscopy.

In addition to the above, other plant tissues can be used as a material for the delivery of nanoparticles, for example, meristems, morphogenic callus, axillary buds, explants, germ, callus, pollen, leaves, anthers, roots, root tips, flowers, seeds, pods and stems. In order to improve delivery efficiency, pretreatment of plant material can be additionally applied (for example, partial maceration of cell walls, incubation of plant material with solutions with enhanced osmotic properties, for example, mannitol solution).

For the coating of chitosan particles, macromolecules can be used that provide targeted delivery to a specific subcellular compartment: cytosol, nucleus, tonoplasts, plastids, etioplasts, chromoplasts, leukoplasts, elioplasts, protein plastics, amyloplasts, chloroplasts and cavities of a two-layer membrane.

The method developed in this application can be used to deliver proteins (bovine serum albumin, BSA; green fluorescent protein, ZFB), small RNAs (tRNAs, guiding (guiding) RNAs, sgRNAs and any others), DNA (plasmid) to cells and tissues of plants encoding SPS) and ribonuclein complexes of the CRISPR-Cas system (Cas9 nuclease together with guiding RNA); for creating plants with increased productivity by delivering nucleic acids encoding specific polypeptides to the plant, for transferring the human interferon alpha or beta gene to the plant to obtain plants resistant to viral infection. The results show that the developed method for the delivery of biological macromolecules to plant cells, based on the infiltration (mechanical or vacuum) of a preparation of chitosan particles or together particles of chitosan and gold with macromolecules immobilized on their surface, ensures efficient delivery of the obtained nanoplatforms, which is confirmed by the identification of labeled fluorescent labels of proteins and nucleic acids in plant cells (Fig. 1), as well as the results of direct sequencing of the next generation in the case of the delivery of specific CRISPR / Cas9 ribonucleoprotein complexes (see examples).

Estimation of the number of edited cells of the next generation in experiments on editing the genome after delivery of the ribonucleoprotein complex of the CRISPR / Cas system to the tissue of plants Nicotiana benthamiana or Solanum tuberosum showed that the editing efficiency when processing samples with Cas9-sgRNA complexes on chitosan nanoparticles is unexpectedly high, not less than 3 5 times, and in some cases 10 times, exceeding that with a similar tissue treatment with Cas9-sgRNA complexes in the absence of nanoparticles, which is an important new technical result.

Experiments to estimate the amount of material delivered by chitosan nanoparticles showed that when using macroinjections or vacuum infiltration for various types of proteins, it is from 1 to 5 mg / ml, for RNA about 15 mm and for DNA about 1 mg / ml, up to 5 mg / ml for nucleoprotein complexes. During the functionalization of the chitosan molecule, there are no restrictions on the structure of the delivered macromolecule, i.e. loading of the nanoparticle can be carried out by any protein of animal, plant or artificial origin currently known, any protein \ DNA or protein \ RNA, DNA or RNA complex of any length and any degree of purification.

The method for delivering macromolecules into plant tissues proposed in the present invention is universal and can be applied to any known agricultural crops (in this list, for example, cereals, legumes, fodder, oilseeds, essential oil, technical, vegetable, medicinal, floral, fruit, berry plants, potatoes, tomatoes, sugar beets, grapes, tobacco, carrots, corn, canola, canola, cotton, palm, peanuts, soybeans, Oryza sp., Arabidopsis sp., Ricinus sp. sugarcane) and ornamental plants.

A further increase in the delivery efficiency of macromolecules, DNA, RNA and proteins is achieved by incubation of plant material in osmotic solutions. For this, 4-20 hours after the infiltration, the treated plant material is incubated in a highly osmotic medium containing up to 500 mOsm / kg of sucrose. Such processing is able to increase the delivery efficiency of macromolecules by another 4-5 times.

Examples

Example 1. Delivery of green fluorescent protein (PBS) into plant cells of Nicotiana benthamiana.

A sample for infiltration was obtained by mixing 1.75 mg / ml of a solution of chitosan in 30 mM Na-acetate buffer (pH 4.5), an aqueous solution of TPF (1 mg / ml) in a volume of 50 and 20 μl, respectively, to form particles of chitosan. then, 15 μl of a 1 mg / ml ZBF solution (TurboGFP, Eurogen) was added to the sample and incubated for 15 min. at a temperature of 25 ° C with constant stirring to immobilize the protein on chitosan particles. The delivery of nanoplatforms to plant cells was carried out by mechanical or vacuum infiltration.

In a number of experiments, gold particles (BioRad, Microcarrier Gold 0.6 μm in size) were additionally added to the sample for infiltration. A solution of gold particles with a concentration of 30 μg / ml was used. Sounding of gold particles was preliminarily performed at a temperature of 25 ° С for 10 minutes with ultrasound at a frequency of 50/60 Hz using an Elmasonic s300h ultrasonic cleaner (Germany). A sample for infiltration was obtained by mixing a solution of gold particles with a concentration of 30 μg / ml, a 0.5% solution of chitosan particles prepared according to the above method, and a solution of ZBF (TurboGFP, Eurogen with a concentration of 1 μg / ml) in a ratio of 40, 140 and 30 μl, respectively. The sample was incubated for 15 minutes. at a temperature of 25 ° C with constant stirring. The delivery of nanoplatforms to plant cells was carried out by mechanical or vacuum infiltration. No visible difference in the delivery efficiency by using chitosan particles was found with or without adding gold nanoparticles, which indicates approximately the same efficiency of the methods. A phase-contrast image of chitosan particles during various processing, obtained using a fluorescence microscope, is shown in FIG. one.

Example 2. Delivery of bovine serum albumin labeled with FITC into plant cells of Nicotiana benthamiana and Solanum tuberosum.

Labeling of bovine serum albumin (BSA) FITC was preliminarily carried out. For this, just before labeling, a BSA solution was prepared at a concentration of 5 mg / ml in 0.1 M carbonate buffer with a pH of 9.0, and 50 μl of the initial FITC solution (Sigma) was added for each 1 ml of protein, then ammonium chloride was added to the solution (up to final concentration of 50 mM) and left overnight at 4 ° C in the dark. Labeled FITC BSA was separated from free FITC by gel filtration on Sephadex G-50 (Pharmacea). The labeling efficiency of BSA FITC was calculated using the appropriate formula, the determined molar ratio of FITC / BSA was 1.5. An infiltration sample was prepared by mixing a solution of chitosan particles obtained according to the above procedure (1.75 mg / ml of a solution of chitosan in 30 mM acetate buffer (pH 4.5), a solution of TPF (1 mg / ml) in a ratio of 100 to 40 μl) and 100 μl of a BSA / FITZ solution (concentration of 3 mg / ml). The sample was incubated for 15 minutes. at a temperature of 25 ° C with constant stirring. Nanoplatforms were delivered to plant cells by mechanical (macroinjection with a syringe) or vacuum infiltration.

In a number of experiments, gold particles (BioRad, Microcarrier Gold 0.6 μm in size) were additionally added to the sample for infiltration. A solution of gold particles with a concentration of 30 μg / ml was used. Sounding of gold particles was preliminarily performed at a temperature of 25 ° С for 10 minutes with ultrasound at a frequency of 50/60 Hz using an Elmasonic s300h ultrasonic cleaner (Germany). An infiltration sample was prepared by mixing 40 μl of a solution of gold particles with a concentration of 30 μg / ml, 140 μl of a 0.5% solution of chitosan particles prepared according to the above procedure, and 100 μl of a solution of FITZ-BSA with a concentration of 3 mg / ml. The sample was incubated for 15 minutes. at a temperature of 25 ° C with constant stirring. The delivery of nanoplatforms to plant cells was carried out by mechanical or vacuum infiltration. No visible difference in the delivery efficiency by using chitosan particles with and without the addition of gold nanoparticles was found, which indicates approximately the same efficiency of the methods (Fig. 2).

Example 3. Delivery of small RNA labeled with a fluorescent label Su-3, in plant cells Nicotiana benthamiana.

In the experiments, preparations of tRNA or synthesized in vitro (as described below, hydraulic RNA) were used. Small RNAs were preliminarily labeled with fluorescent dye Cu3 (Jena Bioscience) using a standard technique. An infiltration sample was prepared by mixing a solution of chitosan particles obtained according to the above procedure (1.75 mg / ml of a solution of chitosan in 30 mM acetate buffer (pH 4.5), a solution of TPF (1 mg / ml) in a ratio of 100 to 40 μl) and 30 μl of a solution of hydraulic RNA (concentration of 15 mM) or tRNA preparation (concentration of 1 mg / ml). The sample was incubated for 5 minutes. at a temperature of 4 ° C with constant stirring. The delivery of nanoplatforms to plant cells was carried out by mechanical or vacuum infiltration. The penetration of tRNA into a plant cell is shown in FIG. 3.

Example 4. Delivery of a plasmid (DNA) encoding a green fluorescent protein into Nicotiana benthamiana plant cells.

Plasmid DNA from cells of E. coli strain XL-blue was isolated by the standard method using a Plasmid Miniprep kit (Eurogen) at a concentration of not less than 1 μg / μl. An infiltration sample was prepared by mixing a solution of chitosan particles obtained according to the above procedure (1.75 mg / ml of a solution of chitosan in 30 mM acetate buffer (pH 4.5), a solution of TPF (1 mg / ml) in a ratio of 100 to 40 μl) and 20 μl of DNA solution. The sample was incubated for 15 minutes. at a temperature of 25 ° C with constant stirring. The delivery of nanoplatforms to plant cells was carried out by mechanical or vacuum infiltration. In FIG. 4, effective fluorescence (expression of the ZFB gene) is observed in plant cells after infiltration with a plasmid containing the ZFB gene, compared with the control plasmid (without the ZFB gene).

Example 5. Editing the coilin gene to produce plants with increased resistance to the Y potato virus.

Editing was carried out by delivery of the CRISPR / Cas system ribonucleoprotein complex, including the guide (hydrofluoric) RNA and S. pyogenes nuclease Cas9 into Nicotiana benthamiana or Solanum tuberosum plant cells.

To obtain the ribonucleoprotein complex, EnGen® Cas9 NLS, S. pyogenes, manufactured by England Biolabs, and hydrotoxic RNAs synthesized using the Invitrogen ™ MEGAscript® T7 Transcription Kit were used. RNPs were prepared by mixing 1 μl of Cas9 nuclease with an initial concentration of 20 μM, 2 μl of guiding RNA (15 μM concentration), 3 μl of 10-fold Cas9 buffer (10X Cas9 Nuclease Reaction Buffer), and 14 μl of sterile H ₂ O nuclease free. The sample was incubated for 10 minutes. at room temperature and mixed with 80 μl of a pre-voiced solution of gold particles or with a solution of chitosan particles obtained as described above. As a control, 80 μl of H ₂ O was added to the sample. The delivery of nanoplatforms to plant cells was carried out by mechanical or vacuum infiltration.

The editing of the coilin gene was evaluated by the next generation high-performance sequencing method: a deletion of about 70 nucleotides in size of the proposed cutting site was detected in the Solanum tuberosum coilin gene.

Example 6: Sequencing of an Edited Potato Coilin Gene

Targeted Amplification:

Amplification of the genomic editing region of apical meristems subjected to coilin gene editing was carried out using primers F 5'-TGATGTTCCCTTCTGCTGTAGGTTGGGAA-3 'and R 5'-GCGTGGTTTTGTATTGAGAAGAGTCATA-3'. These primers were designed with the ability to identify not only point mutations, but also large deletions. Amplification was carried out in 50 μl of the mixture using DreamTaq PCR Master Mix 2x (Thermo Scientific, USA) with the following parameters: primary denaturation at 94 ° C for 2 minutes; 40 cycles: 94 ° C, 65 ° C and 72 ° C each cycle for 40 seconds. Amplification products were purified using Agencourt AMPure beads (Beckman Coulter, USA)

Library Preparation and Sequencing:

Preparation of the library for sequencing and sequencing were performed in accordance with the instructions from the equipment manufacturer (Thermo Scientific, USA). The concentration of the library for sequencing was determined using the Ion Library TaqMan ™ Quantitation Kit (Thermo Scientific, USA). An additional quality control was electrophoresis. As a result, 2 fragments of ≈360 and 300 nucleotide pairs were identified (Fig. 5).

The matrix was prepared and loaded onto the chip with Ion Chef (Thermo Scientific, USA) using the reagents Ion 520 ™ & Ion 530 ™ Kit-Chef (Thermo Scientific, USA) and Ion 520 ™ Chip Kit (Thermo Scientific, USA). Sequencing was performed on an Ion S5 instrument (Thermo Scientific, USA).

Processing sequence data:

The resulting reads were subjected to secondary processing, sequencing data was filtered by QV≥20 quality and reads less than 200 nucleotides in length were removed. Mapping was performed on the wild-type sequence (WT) using a standard protocol. Alignments are shown in FIG. 6.

Example 7: Quantification of the effectiveness of editing the genome of plants.

Editing efficiency was evaluated in the experiment on editing the coilin gene after delivery of the ribonucleoprotein complex of the CRISPR / Cas system to the plant tissue, including the guiding RNA and S. pyogenes nuclease Cas9 into Nicotiana benthamiana or Solanum tuberosum plant cells. Efficiency was estimated as a percentage as the ratio of the number of edited sequences (loci) (with the edited coilin gene) to unedited (having the original gene sequence). To do this, DNA is extracted from the tissues of nanoparticle-treated cells / apical meristems / axillary buds / whole plants, and then, as described in Example 6, targeted sequencing of the target DNA section subjected to editing is carried out. After that, the number of readings (nucleotide sequences) containing directed mutations is compared with readings whose nucleotide sequences do not contain such mutations. The ratio of the number of reads containing editing acts to the total number of reads gives the effectiveness of genomic editing.

When processing gold nanoparticles coated with Cas9-sgRNA complexes, and during vacuum or mechanical infiltration with Cas9-sgRNA complexes on chitosan nanoparticles, the transformation efficiency was in the range of 10-20%. Processing by vacuum or mechanical infiltration with Cas9-sgRNA complexes without nanoparticles (- control) showed delivery efficiency in the range of 0-5%.

Example 8. Genomic editing of morphogenic potato calli using chitosan nanoparticle infiltration.

To obtain morphogenic calluses and regenerated plants, stem segments (0.5-1.0 cm) were cut from potato plants in vitro and cultivated in petri dishes for 21 days on Murashige and Skoog medium containing a set of vitamins according to Murashige and Skoog (2 mg / l glycine, 0.5 mg / l nicotinic acid, 0.5 mg / l pyridoxine НСl, 0.4 mg / l thiamine НСl), 100 mg / l inositol, 30 g / l sucrose, 7 g / l agar, pH 5.8, and Zeatin phytohormones 3 mg / l , NAA 2 mg / L, GA ₃ 1 mg / L. The explants were cultured under lighting conditions (16 hours 40 μmol m ^-2 s ¹ ) at 22-25 ° C. The formed morphogenic calli were separated from stem explants (shown in Fig. 7) and immersed in a suspension of particles. Chitosan nanoparticles were infiltrated with the Cas9-sgRNA complex immobilized on their surface to the phytoendesaturase gene by evacuation at ~ 0.9 bar, after which the calli were transferred to the shoot regeneration medium mentioned above. 21 days after the infiltration, the calli forming shoots (Fig. 8, A) were transferred to fresh Murasige and Skoog medium without phytohormones (Fig. 8, B) for the further development and growth of shoots and their analysis. Genomic editing was confirmed using the T7E1 endonuclease test, which was carried out according to the standard method described on the website of the manufacturer of endonuclease (https://www.neb.com/protocols/2014/08/11/determining-genome-targeting-efficiency-using- t7-endonuclease-i). Test results were detected by agarose gel electrophoresis. On average, one in 10 morphogenic calli demonstrated the presence of genomic editing at the desired locus (Fig. 8).

Claims (21)

1. A method for the delivery of macromolecules selected from nucleic acids, proteins or nucleoprotein complexes to a plant cell, where the method includes: ensuring the presence of a plant cell having a cell wall; coating the surface of a chitosan nanoparticle with a size of 600-800 nm with a nucleic acid, protein or nucleoprotein complex; bringing a plant cell having a cell wall and said coated nanoparticle into contact with each other; and providing absorption of the specified nanoparticles in the plant cell containing the cell wall. 2. The method according to p. 1, in which the coating of the nanoparticles includes the immobilization of a macromolecule due to non-covalent adsorption on the surface of the nanoparticles. 3. The method of claim 1, wherein the nanoparticle is a multifunctionalized nanoparticle. 4. The method of claim 1, further comprising absorbing the macromolecule into the nanoparticle. 5. The method according to any one of paragraphs. 1-4, which further includes providing the absorption of the nanoparticle compartment of the plant cell. 6. The method according to any one of paragraphs. 1-4, which further includes coating the nanoparticle with a peptide penetrating the cell and / or a protein that provides targeted delivery to the subcellular compartment. 7. The method according to any one of paragraphs. 1-6, where the nanoparticles are introduced into the plant cell using macroinjection, biolistics or vacuum. 8. The method according to any one of paragraphs. 1-7, in which the nanoparticles are used for delivery to the cell of RNA, DNA and ribonucleic complexes. 9. The method according to p. 8, where the RNA is selected from tRNA, guide RNA, subgenomic RNA. 10. The method according to p. 8, where the ribonucleic complexes are related to the CRISPR-Cas system. 11. The method of claim 8, wherein the ribonucleic complex comprises Cas9 nuclease in association with guiding RNA. 12. The method according to any one of paragraphs. 1-11, which further includes the selection of edited cells. 13. The method according to p. 12, in which the selected cells are regenerable cells. 14. The method according to p. 12, where the selection is carried out using genetic analysis methods. 15. The method according to p. 12, where the selection is carried out using PCR-RV and / or sequencing methods. 16. The method according to any one of paragraphs. 1-15, where delivery is carried out in plant tissue selected from meristems, morphogenic callus, axillary buds, explants, embryos, callus, pollen, leaves, anthers, roots, root tips, flowers, seeds, pods and stems. 17. The method according to any one of paragraphs. 1-16, where the pretreatment of plant material is additionally applied by incubation with solutions with enhanced osmotic properties.

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