KR101900465B1 - Technology to deliver the genome editing tool by using the CRISPR-CAS family - Google Patents

Abstract

The present invention relates to a method for producing a large amount of exosome (Cas9: EXPLOR) containing Cas9 protein, a vector for producing exosome that can be used for producing the exosome, an exosome containing Cas9 protein prepared by the above method, ≪ / RTI > and a DNA sequencing composition containing the same. Since the exosome containing Cas9 protein can be produced with high yield by using the method for producing exosome including Cas9 protein provided by the present invention, Can be widely used in the delivery technology of the system.

Description

[Technical Field] The present invention relates to a genome editing tool using CRISPR-CAS family,

The present invention relates to a method for producing an exosome comprising a Cas9 protein using a photo-specific binding protein, and a DNA sequence control composition containing an exosome prepared by the above-mentioned method as an active ingredient.

The human body is composed of about 200 kinds of 100 trillion cells, and its bioactivity is regulated by the action of various proteins in the cell to maintain its life.

Cells are surrounded by a bilayer membranous membrane composed of phospholipids, which blocks the entry of foreign substances into cells. Most of the protein therapeutics developed so far also pass through the cell membrane and can not enter the cytoplasm. They have physiological effects by acting outside the cell or by acting on a receptor on the cell membrane to transmit signals into the cell.

There are numerous proteins in the cytoplasm, which interact with each other to regulate their physiological activity. If the protein therapeutic can be put directly into the cell, that is, into the cytoplasm, the cell activity will be controlled more effectively.

Recently, studies on the direct introduction of the target protein into the cell through the cell membrane have been actively conducted. Protein Transduction Domains (PTDs), which are peptides that pass through the cell membrane, and recombinant proteins of the target protein can be prepared and introduced into the cytoplasm through the cell membrane (FIG. 1). The PTDs include HIV-1 TAT, HSV VP22, Antp, dfTAT, and Hph-1. Fusion proteins that bind these proteins with target proteins need to be produced and separated in the form of recombinant proteins. There is a problem in that refolding of the protein is not performed properly, the activity is decreased, the protein is nonspecifically transmitted, the risk of causing an immune reaction in vivo is large, the cost is high, and the yield is low.

The target protein bound to various nanoparticles can enter the cytoplasm through the cell membrane by endocytosis (Fig. 2). The nanoparticles include Gold NP, Liposome NP, Magnetic NP, and Polymeric NP. Most of the degradation products of these proteins and proteins bind to lysosomes in the cell, , It is difficult for the target protein and the nanoparticles to separate from each other in the cytoplasm, and the toxicity of the nanoparticles may become a problem.

Exosome refers to a small vesicle of membrane structure with a size of 50 - 200 nm that is secreted out of the cell with protein, DNA, RNA, etc., for intercellular signaling.

Exosomes were originally found in the process of leaving hemoglobin in the red blood cells by eliminating and exiting the intracellular proteins at the last stage when the red blood cells were matured. These exosomes were found in the electron microscopic study, But rather from a specific compartment within the cell called Multivesicular bodies (MVBs), which is released and released outside the cell. That is, when fusions between the polycation and the plasma membrane occur, such follicles are released into the extracellular environment, which is called exosomes (FIG. 3).

Although it is unclear how these exosomes are produced by any molecular mechanism, it is possible to identify a variety of immune cells and stem cells, including B-lymphocytes, T-lymphocytes, dendritic cells, megakaryocytes, macrophages, Tumor cells are also known to produce and secrete exosomes in a living state.

Exosomes contain various proteins, DNA, and RNA in the cell. The substances contained in the exosome and secreted out of the cell may be re-introduced into other cells by fusion or endocytosis with the cell membrane and serve as intercellular communication partners. In addition, the substances contained in the exosome and secreted out of the cell can be analyzed and used for diagnosis of a specific disease.

Several types of microRNAs are contained in exosomes, and a method for diagnosing disease by detecting the presence or abundance thereof is known (see KR 10-2010-0127768A). In particular, WO2009-015357A discloses a method for detecting exosomes present in a cancer-derived sample (blood, saliva, tears, etc.) and measuring the amount of microRNA change. When the increase or decrease is compared with the control, And a method for diagnosing the abnormality is disclosed. Particularly, exosomes obtained from patients having a specific disease (lung disease) are analyzed to specifically describe the relationship between a specific microRNA and lung disease. Also, in addition to pulmonary disease, methods for diagnosing renal disease using protein contained in exosome have been studied.

Exosomes also contain antigens. On the other hand, in antigen presenting cells (APCs), antigen peptides are loaded on MHC (major histocompatibility complex) class II molecules in intracellular compartments containing membranes, including polycations. Therefore, Peptide-MHC class II complex. Thus, exosomes can present antigen peptides to CD4 + T lymphocytes as transporters of immunogens, thereby inducing an immune response such as proliferation of T lymphocytes. In addition, since exosomes are enriched with molecules capable of stimulating an immune response, such as MHC class I, heat-shock proteins (HSPs), they are immune to autoimmune disease or tumor treatment May be used for the purpose of enhancing or reducing.

In addition, the exosome in which the target protein is contained can be used for the treatment of various diseases in vivo. For example, a protein exhibiting an anti-cancer effect as a target protein, or an exosome containing siRNA can be prepared, and then treated with cancer cells to treat cancer (FIG. 4).

Thus, exosomes containing the desired protein can be used for the treatment of disease. For this purpose, it is necessary to be able to efficiently produce exosome containing the target protein. Korean Patent Publication No. 2004-0015508 describes a method for producing an exosome comprising a specific antigen. There is disclosed a method in which a gene for a specific antigen is introduced into a cell line and the protein of the introduced gene is stably expressed in the cell line and is released to the outside of the cell through the exosome and a method of using such exosome as a vaccine .

However, since the exosome is formed naturally in the cell, even if a gene encoding the target protein is introduced into the cell that produces the exosome, the exosome in which the expressed protein is contained can be produced Is very low.

Accordingly, the present inventors have made efforts to develop a method for more effectively producing an exosome containing a target protein. As a result, they have found that a fusion protein obtained by fusing an exosome-specific marker and a target protein is expressed in a cell producing a large amount of exosome , It was confirmed that the exosome containing the target protein can be efficiently produced (Fig. 5).

In addition, in the above method, the target protein is attached to the membrane of the exosome, and a fusion protein obtained by fusing each of the light-specific binding protein pairs to each of the exosome-specific marker and the target protein is called a cell that produces a large amount of exosome When these fusion proteins are introduced into the exosome by the exosome-specific marker and the irradiation of the light is stopped after the introduction of the exosome-specific marker, the exosome- It was confirmed that the fusion protein of the red binding protein was isolated and the exosome containing the target protein in the free state could be effectively produced (FIG. 6).

CRISPR-Cas9 is an RNA-based artificial restriction enzyme that is a complex of RNA and protein that cleaves a specific part of a gene to enable genetic modification. Recently, it has been widely recognized as a genetic engineering material.

CRISPR is an abbreviation for Clustered regularly-interspaced short palindromic repeats. It is a kind of repetitive gypsum sequence and is the first acquired bacterial acquisition immune system. When the virus enters the cells of the bacterium, the Cas9 protein first recognizes and cleaves. Thereafter, the viral sequence inserted into the crystal sequence is inserted, and the RNA in which the crystal sequence and the viral sequence are combined is transcribed. This then serves as an RNA that forms the Cas9 complex. When the same virus is invaded, the transferred 'Crisp + virus sequence' is complexed with Cas9 to remove the virus more rapidly than Cas9 alone. Using this principle, a specific base sequence is attached to the Cas9 complex, and the Cas9 protein cuts the designated base sequence, which can be applied to genetic engineering.

Cpf1 is a protein that exhibits similar effects as Cas9 of the engineered endonuclease system CRISPR-Cas9. As described below, Cpf1 is a PAM sequence different from Cas9 (Protospacer Adjacent Motif: all the gene scissors must approach one end of the target DNA to start DNA cleavage, and the short gene sequence, PAM of Cpf1: TTTC ; PAM of Cas9: NGG) and can be used in areas that Cas9 does not recognize. In particular, short crRNA (Crispr RNA, which binds to target DNA and Cpf1 can cause DNA cleavage) It is a protein that works and can be used more practically. In the case of Cas9, tracrRNA is additionally required.

[Figure 1] Comparison of Cas9 protein and Cpf1 protein

The present inventors have confirmed that Cas9 protein is transferred to the cytoplasm of the target cell using exosomes containing Cas9 or Cpf1 protein internally. The present inventors confirmed that the Cas9 protein is transferred to the cytoplasm of the target cell, and the delivery of the exosome genome editing system And confirmed the possibility as a technology, thereby completing the present invention.

It is an object of the present invention to provide a method for mass production of exosome comprising a fusion protein of Exosome-specific marker and Cas9 protein.

It is another object of the present invention to provide a method for mass-production of exosome containing Cas9 protein isolated from exosome membrane using a photo-specific binding protein pair.

Another object of the present invention is to provide a vector for producing an exosome which can be used for the production of the exosome.

It is another object of the present invention to provide a DNA sequence manipulating composition comprising the exosome and a guide RNA (gRNA) specific to the target DNA sequence.

According to an aspect of the present invention,

a) obtaining a transformed cell by introducing into the exosome-producing cell a polynucleotide encoding a fusion protein in which a CasS protein is bound to an exosome-specific marker; And b) culturing the transformed cells. The present invention provides a method for mass-producing exosomes attached to exosome membranes.

In addition,

a) a polynucleotide encoding a fusion protein (first fusion protein) in which the exosome-specific marker and the first photo-specific binding protein are bound to the exosome-producing cell, and a polynucleotide that binds to the first photo- Introducing a polynucleotide encoding a fusion protein (second fusion protein) in the form of a Cas9 protein-bound second photo-specific binding protein; b) irradiating the exosome-producing cell with light capable of inducing binding of the first photo-specific binding protein and the second photo-specific binding protein; And c) stopping the irradiation of the exosome after the exosome is produced in the exosome-producing cell. The present invention also provides a method for mass-producing exosome comprising Cas9 protein.

In addition,

(a) a first expression vector comprising a polynucleotide encoding a fusion protein (first fusion protein) in which a first light-specific binding protein and an exosome-specific marker are bound; And (b) a polynucleotide capable of introducing a polynucleotide encoding the Cas9 protein and a polynucleotide encoding a second photo-specific binding protein capable of binding the first photo-specific binding protein. A vector for producing an exosome, which comprises an expression vector.

In addition, the present invention provides a method for delivering Cas9 protein to a cytoplasm, which comprises using an exosome containing Cas9 protein, which is produced using the above method, and the exosome, And DNA sequencing compositions comprising specific guide RNA (gRNA).

The exosome comprising Cas9 protein can be produced with high yield by using the method of producing exosome including Cas9 protein provided by the present invention and because Cas9 protein is separated from exosome membrane, It can be widely used as a DNA sequence control composition using cotton.

FIG. 1 is a diagram showing a cytoplasmic delivery method of a target protein through a cell membrane transduction domain (PTD) and a recombinant protein of a target protein (Steven R. et al., Protein transduction: unrestricted delivery into all cells, Trends in Cell Biology, .
FIG. 2 is a graph showing the intracellular delivery of a target protein through endocytosis to a conjugate comprising nanoparticles and a target protein (Munish Chanana et al., Physicochemical properties of protein-coated gold nanoparticles in biological fluids and cells before and after proteolytic digestion, Angew, Chem., Int.
FIG. 3 is a view showing the process in which exosomes are released from the cells and released from the cells of the multi-vesicular bodies (MVBs) (Grace Raposo and Willem Stoorvogel, Extracellular vesicles: Exosomes, microvesicles, and friends. (4), 373-383, 2013).
FIG. 4 is a diagram showing a process of delivering siRNA in vivo through a targeted exosome to treat cancer (Alvarez-Erviti, L. et al. Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nature biotechnology 29, 341-345, 2011).
FIG. 5 is a schematic diagram showing a process for producing protein-carrying exosomes (EXPLORs) designed as optogenetically-designed proteins according to the present invention.
FIG. 6 is a diagram showing a process of separating the fusion protein of the target protein and the light-specific binding protein from the inside of the exosome when the light irradiation of EXPLORs according to the present invention is stopped.
FIG. 7 is a fluorescence photograph showing the change in the intracellular location of mCherry protein in a transformant into which CIBN-EGFP-CD9 gene and mCherry-CRY2 gene are introduced into HEK293T cells according to irradiation of blue light.
8 is a diagram showing an experimental procedure for obtaining EXPLORs according to the present invention.
FIG. 9 is an immunoblot analysis image showing the result of measurement of a change in the content of a target protein (mCherry protein) captured inside the exosome according to the intensity of blue light.
FIG. 10 is an electron micrograph showing the result of treating target cells (HT1080 cells) treated with exosome containing a target protein (mCherry) and confirming the introduction of a target protein into the target cells. And the right side represents the target cell treated with exosome.
FIG. 11 is a graph showing the fluorescence image (a) showing the result of examining whether the target protein is introduced into the target cell (HT1080 cell) after treating the exosome containing the target protein (mCherry) (B) showing the results of comparing the ratios of the apoptotic cells induced by the apoptotic cells.
FIG. 12 is a fluorescence image showing the change in intracellular location of mCherry protein according to whether blue light was irradiated in a transformant into which GIGANTEA-EGFP-CD9 gene and mCherry-FKF1LOV were introduced into HEK293T cells.
13 shows the degree of expression (a) of luciferase-mCherry fusion protein using fluorescence imaging and the number of luciferase activity and the number of molecules in the produced cells (b):
Control: HEK293T cells without any treatment;
OVER: HEK293T cells transfected with Luciferase-mCherry-CRY2 alone;
XP: HEK293T cells transfected with XPACK-Luciferase-mCherry using XPACK (Systems Biosciences), a commercial vector designed for exosome loading technology;
EXPLOR: HEK293T cells transfected with Luciferase-mCherry-CRY2 and CIBN-EGFP-CD9 according to the method of the present invention.
Fig. 14 is a diagram showing the measurement of the luciferase activity (a) and the number of molecules (b) in the produced exosome:
NEG: Exosomes produced in HEK293T cells that were not treated with anything;
OVER: Exosomes produced in HEK293T cells transfected with Luciferase-mCherry-CRY2;
XP: Exosomes produced in HEK293T cells using XPACK (Luciferase-mCherry XPACK) using XPACK (Systems Biosciences), a commercial vector designed for exosome loading technology;
EXPLOR: Exosomes produced in HEK293T cells transfected with Luciferase-mCherry-CRY2 and CIBN-EGFP-CD9 according to the present invention;
ON: Exosomes produced by culturing in 200 μW of blue light for 72 hours,
OFF: Exosome produced by culturing for 72 hours in the absence of light.
FIG. 15 is a diagram showing the loading efficiency of a target protein in the produced exosome. FIG.
FIG. 16 shows the efficiency of target protein transfer to target cells (HeLa) using exosomes:
Control: Exosomes produced in HEK293T cells without any treatment;
OVER: Exosomes produced in HEK293T cells transfected with Luciferase-mCherry-CRY2;
XP: Exosomes produced in HEK293T cells using XPACK (Luciferase-mCherry XPACK) using XPACK (Systems Biosciences), a commercial vector designed for exosome loading technology;
EXPLOR: Exosomes produced in HEK293T cells transfected with Luciferase-mCherry-CRY2 and CIBN-EGFP-CD9 according to the present invention;
ON: Exosomes produced by culturing in 200 μW of blue light for 72 hours,
OFF: Exosome produced by culturing for 72 hours in the absence of light.
FIG. 17 shows that Luciferase-mCherry-CRY2 and CIBN-EGFP-CD9 are expressed in the same position in HEK293T cells.
FIG. 18 shows that Cas9-mCherry-CRY2 and CIBN-EGFP-CD9 are expressed at the same position in HEK293T cells in the cells.
19 is a diagram showing a plasmid vector for producing exosome (Cas9: EXPLOR) containing Cas9 protein.

Hereinafter, the present invention will be described in detail.

The present invention relates to a method for efficiently and mass-producing an exosome containing Cas9 protein.

The present inventors focused on exosome-specific markers (CD9, CD63, CD81 or CD82) while carrying out various studies in order to develop a method for more effectively producing exosomes containing the target protein therein. Since the exosome-specific marker has a common property that it is a membrane protein of 4-fold penetration type belonging to the tetraspanin family, when the desired protein is bound to the membrane protein of the exosome, it can be relatively easily contained in the exosome Respectively.

The exosome-specific marker that is specifically present in the exosome membrane, which is specifically present in the exosome membrane, and the exosome-specific marker and the fusion protein of the target protein are expressed in a cell that mass-produces the exosome, a large amount of the exosome containing the target protein can be produced .

Specifically, a method for producing an exosome comprising a Cas9 protein, which is a target protein of the present invention, comprises expressing an exosome-producing cell by introducing a polynucleotide encoding a fusion protein in which an exosome-specific marker and Cas9 protein are bound do.

The exosomes generated at this time are fused with exosome-specific markers in which Cas9 protein is embedded in the exosome membrane.

The target protein is bound to the membrane protein of the exosome and is not separated even after reaching the target cell. In order to solve these problems, various studies have been conducted. As a result, it has been found that, by using a photo-specific binding protein, a technology for temporarily producing an exosome containing the target protein by binding the target protein with the marker protein Respectively. For example, CIBN and CRY2, which are light specific binding proteins, can be used. Specifically, CIBN is expressed in a form fused with CD9, which is one of the marker proteins, and CRY2 is fused with a target protein A gene encoding these fusion proteins was introduced into the exosome-producing cells. The CIBN-CD9 fusion protein expressed in the exosome-producing cell is included in the exosome due to CD9. When the cell is irradiated with blue LED light, the target protein-CRY2 fusion protein expressed in the exosome- Is bound to the CIBN domain fused to CD9, a reversibly derived fusion protein of the target protein -CRY2-CIBN-CD9 form is formed, and such fusion protein is contained in the exosome due to CD9. When the blue light LED illumination is no longer irradiated after the exosome containing the target protein is produced, the CIBN-CRY2 binding is released and the target protein is not bound to the cell membrane of the exosome, , So that an exosome containing the target protein can be produced as a result (FIGS. 5 to 10).

The exosome thus produced may exhibit a completely different effect from the exosome including the conventional target substance. Since the conventional exosome has been expressed in the form of being fused to the exosome-specific marker in order to trap the target protein inside the exosome, the target protein is trapped in the exosome, but is still attached to the exosome membrane The target protein can be transferred to the target cell only when the exosome is fused to the cell membrane of the target cell. Thus, even when the target protein is fused to the target cell, the target protein can be bound to the exosome fused to the exosome membrane , There is a limitation that the target protein has a very low probability of exhibiting its effect in the target cell. However, since the exosome provided in the present invention is trapped in the exosome without the target protein bound to the membrane, when the exosome is endocytosed by the target cell and enters the cytoplasm, the exosome is attached to the exosome membrane Therefore, when the exosome is degraded, the target protein can be transferred to the cytoplasm of the target cell, and the target protein can freely move within the cytoplasm of the target cell, so that the target protein has an advantage that it can sufficiently exhibit the physiological activity in the target cytoplasm ( 11).

In addition, since the binding level of the target protein to be bound to the marker protein is changed according to the intensity of the light irradiated upon light irradiation, the content of the target protein captured in the exosome can be controlled when the light intensity is controlled .

Therefore, a method for producing an exosome containing a target protein using a photo-specific binding protein has not been known at all and has been developed for the first time by the present inventors.

Accordingly, the present invention provides a method for preparing an exosome comprising Cas9 protein comprising the steps of:

(a) a polynucleotide encoding a fusion protein (first fusion protein) in which the exosome-specific marker and the first photo-specific binding protein are bound to the exosome-producing cell, and a polynucleotide encoding the first specific binding protein Introducing a polynucleotide encoding a fusion protein (a second fusion protein) in which a second photo-specific binding protein and Cas9 protein are combined;

(b) irradiating the exosome-producing cell with light capable of inducing binding of the first photo-specific binding protein and the second photo-specific binding protein; And

(c) after the exosome is produced in the exosome-producing cell, stopping the irradiation of the light.

The term "exosome " of the present invention means a small vesicle of a plasma membrane structure originating from a specific compartment within a cell called multivesicular bodies (MVBs) and released or secreted out of the cell.

In the present invention, the exosome may serve as a carrier for transporting a target protein to a target cell or tissue of a target protein by including a target protein therein. The target protein carried by the exosome may be a target cell Or may be used to treat certain diseases or to diagnose certain diseases by acting on the tissues.

The term "exosome-producing cell" of the present invention means a cell capable of producing the exosome.

In the present invention, the exosome-producing cell may be, but not limited to, a B-lymphocyte, a T-lymphocyte, a dendritic cell, a megakaryocyte, a macrophage, a stem cell, . For example, in the example of the present invention, HEK293T cells, which are a kind of immortalized cell line, were used as the exosome-producing cells.

The term "exosome-specific marker " of the present invention means a protein abundantly present in the membrane of the exosome.

In the present invention, the exosome-specific marker may be, for example, CD9, CD63, CD81, CD82, and the like. For example, in the examples of the present invention, CD9 was used as an exosome-specific marker. CD9, CD63, CD81, and CD82 are 4-fold penetration type membrane proteins. When the desired protein is bound to the exosome membrane protein, the desired protein is easily present in the exosome.

The term "photo-specific binding protein" of the present invention is also referred to as a light-oil-type heterodimer forming protein or a photoinduced homodimer forming protein. When light of a specific wavelength is irradiated, it may bind to a different protein to form a heterodimer Refers to a protein capable of binding to a protein or another protein of the same kind to form a homodimer.

In the present invention, the photo-specific binding protein may be, for example, a light oil-releasing duplex forming protein, but not limited thereto. As another example, a cryptochrome-interacting basic-helix-loop-helix protein, CIBN, phytochrome B, phytochrome interacting factor, FKF1, GIGANTEA, CRY (chryptochrome), PHR (phytolyase homolgous) region), and the like.

In particular, when forming a heterodimer, two photo-specific binding proteins (a first photo-specific binding protein and a second photo-specific binding protein) may be used, wherein the first photo-specific binding protein is CIB or CIBN , The second photo-specific binding protein may be CRY or PHR, and when the first photo-specific binding protein is PhyB, the second photo-specific binding protein may be PIF, When the first photo-specific binding protein is GIGANTEA, the second photo-specific binding protein may be FKF1.

For example, in an embodiment of the present invention, CIBN is used as the first photo-specific binding protein, CRY2 is used as the second photo-specific binding protein, and the wavelength of the used light is set to 460 to 490 nm And the intensity of the light was set to 20 to 50 μW.

Meanwhile, in order to confirm the expression of the first fusion protein in which the exosome-specific marker and the first photo-specific binding protein are bound and the position in the cell, marker proteins may be fused together. For example, in an embodiment of the present invention, expression of the first fusion protein in which CIBN and CD9, or GIGANTEA and CD9 are combined, in a form in which the fluorescent protein EGFP is inserted, And to confirm the intracellular location.

The term "target protein" of the present invention means a protein expressed in a form fused with the second photo-specific binding protein to be contained in the exosome.

As used herein, the term "Cas protein " refers to a protein element essential in the CRISPR / Cas system and to complex with two RNAs, called CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) , An active endonuclease or nickase is formed.

In the present invention, the Cas protein may be any Cas protein if it has endo-nuclease or nicase activity when it forms a complex with the guide RNA. Preferably, the Cas protein is a Cas9 protein or a variant thereof.

In the present invention, the target protein is a Cas9 or Cpf1 protein, and the Cas9 protein is preferably composed of the amino acid sequence shown in SEQ ID NO: 1, but not limited thereto, and the Cpf1 protein has an amino acid sequence represented by SEQ ID NO: 2 But it is not limited thereto.

The term "cultivation" of the present invention means a method of growing a cell or a microorganism under an appropriately artificially controlled environmental condition.

In the present invention, the transformant is cultivated for 1 to 3 days, then replaced with a medium not containing fetal bovine serum, and further cultured for 2 to 5 days.

In the present invention, the method for culturing the transformant may be carried out by a method well known in the art.

The medium means a known medium used for animal cell culture and may be selected from a group such as commercially available serum free media, protein free media and chemically defined media .

Preferably, the serum-free medium is SFM4CHO (HyClone), EX-Cell (JHR Bioscience) or the like, and the insulin-like growth factor I -I), ethanolamine, ferric chloride, and phosphatidyl choline may be added, but the present invention is not limited thereto.

The animal cell culture medium in which the protein derived from animal, in particular, the protein having a molecular weight of 10 kDa or more is removed in the serum-free medium from which the bovine serum component has been removed, is selected as Prostate (Lonza) and PF-CHO (HyClone) But is not limited thereto.

The chemically defined medium is an animal cell culture medium having no known animal-derived components and all constituent components of which are known chemical structures. These media include CDM4CHO (HyClone), PowerCHO2CD (Lonza), and CD-optiCHO ) May be selected, but the present invention is not limited thereto.

The term "first fusion protein" of the present invention means a fusion protein in which the exosome-specific marker and the first photo-specific binding protein are bound.

In the present invention, the sequence of the exosome-specific marker and the first photo-specific binding protein contained in the first fusion protein is such that the first fusion protein binds to the first light in the exosome- As long as it can be expressed so that the specific binding protein can be expressed so as to be positioned so that the N-terminal of the first photo-specific binding protein is bound to the C-terminus of the exosome-specific marker .

The exosome-specific marker constituting the first fusion protein and the first photo-specific binding protein may be directly linked to each other, or may be linked through a linker. The linker is not particularly limited as long as the first fusion protein can be expressed so that the first photo-specific binding protein is located in the exosome-producing cell in the inside of the exosome-producing cell, but a peptide linker composed of an amino acid can be used And more preferably a flexible peptide linker can be used. The peptide linker may be operably linked to an expression vector by linking the nucleic acid encoding the linker in-frame between the nucleic acids encoding each domain.

The term "second fusion protein" of the present invention means a fusion protein in which the second photo-specific binding protein and the target protein are combined.

In the present invention, the order of arrangement of the second photo-specific binding protein and the target protein contained in the second fusion protein is such that the second fusion protein is the first photo-specific of the first fusion protein in the exosome- As long as it can bind to the binding protein site and be located within the exosome, it is not particularly limited. For example, the N-terminal of the target protein is bound to the C-terminus of the second photo- specific binding protein .

In addition, the second photo-specific binding protein and the target protein constructing the second fusion protein may be directly coupled to each other, or may be connected to each other through a linker. The linker is not particularly limited as long as the second fusion protein can be located in the exosome by binding to the first photo-specific binding protein region of the first fusion protein in the exosome-producing cell, Linkers can be used, and more preferably, flexible peptide linkers can be used. The peptide linker may be operably linked to an expression vector by linking the nucleic acid encoding the linker in-frame between the nucleic acids encoding each domain.

In addition, each of the fusion proteins may include a polypeptide having a sequence that differs from a wild-type amino acid sequence of each domain contained therein by one or more amino acid residues. Amino acid exchange in proteins and polypeptides that do not globally alter the activity of the molecule is known in the art. The most commonly occurring exchanges involve amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly. In addition, the protein may include a protein having increased structural stability or increased protein activity due to mutation or modification of the amino acid sequence, such as heat, pH and the like.

Finally, the polypeptide of each domain constituting the fusion protein or the fusion protein may be prepared by a chemical peptide synthesis method known in the art, or may be prepared by amplifying a gene encoding the domain by PCR (polymerase chain reaction) Followed by expression and cloning into an expression vector.

On the other hand, each fusion protein can be expressed in the exosome-producing cell by introducing a polynucleotide encoding the fusion protein into the exosome-producing cell. As the method for introducing the polynucleotide into the exosome-producing cell, Method can be used. For example, a method of introducing using an expression vector can be used.

The term "expression vector" of the present invention refers to a recombinant vector capable of expressing a target peptide in a desired host cell, and a gene product containing an essential regulatory element operatively linked to the expression of the gene insert. The expression vector includes expression control elements such as an initiation codon, a termination codon, a promoter, an operator, etc. The initiation codon and termination codon are generally regarded as part of the nucleotide sequence encoding the polypeptide, and when the gene product is administered, And must be in coding sequence and in frame. The promoter of the vector may be constitutive or inducible.

The term "operably linked" of the present invention means a state in which a nucleic acid sequence encoding a desired protein or RNA is functionally linked to a nucleic acid expression control sequence so as to perform a general function . For example, a nucleic acid sequence encoding a promoter and a protein or RNA may be operably linked to affect the expression of the coding sequence. The operative linkage with an expression vector can be produced using gene recombination techniques well known in the art, and site-specific DNA cleavage and linkage can be performed using enzymes generally known in the art.

In addition, the expression vector may include a signal sequence for the release of the fusion polypeptide to facilitate the separation of the protein from the cell culture medium. A specific initiation signal may also be required for efficient translation of the inserted nucleic acid sequence. These signals include the ATG start codon and adjacent sequences. In some cases, an exogenous translational control signal, which may include the ATG start codon, should be provided. These exogenous translational control signals and initiation codons can be of various natural and synthetic sources. Expression efficiency can be increased by the introduction of suitable transcription or translation enhancers.

According to a preferred embodiment of the present invention, the expression vector can be expressed by binding a tag to the target protein to confirm whether the target protein is inserted into the exosome. Since the tag is for confirming the presence or absence of the target protein, the target protein can be bound to the opposite site to which the second photo-specific binding protein is bound. For example, fluorescence of a red fluorescent protein, The protein can be used as a tag to bind to the C-terminal region of the target protein.

It is possible to confirm whether or not the exosome contains the target protein by expressing the designed protein in the exosome-producing cell and confirming whether the fluorescent protein tag is detected in the exosome after the exosome is produced .

The term "light" of the present invention means light irradiated to temporarily bind the first light-specific binding protein and the second light-specific binding protein expressed in the exosome-producing cell.

As described above, in the exosome-producing cell, the first photo-specific binding protein is expressed in the form of the first fusion protein together with the exosome-specific marker, and the second photo-specific binding protein is expressed in the form of the second fusion protein Specific binding protein is irradiated with light necessary for binding of the first specific binding protein and the second specific binding protein to the exosome-producing cell, the first specific binding protein and the second specific Binding protein is bound and consequently a fusion protein complex having the shape of the exosome-specific marker-first light-specific binding protein-second light-specific binding protein-target protein is temporarily formed. The exosome-producing cell , The target protein may be linked to the exosome due to the exosome-specific marker. In this case, the target protein is present inside the exosome, and when irradiation of light is stopped after the exosome is produced, the first photo-specific binding protein and the second photo-specific binding protein are separated, The target protein present in the inside is released to the outside in the form contained in the exosome upon the release of the exosome. It is also preferable that the light is intermittently irradiated rather than continuously irradiated so that the target protein can be more efficiently introduced into the exosome. That is, when light is intermittently irradiated, the binding and separation of the first photo-specific binding protein and the second photo-specific binding protein are repeated, so that the probability that the target protein is introduced into the exosome can be improved .

Meanwhile, since the wavelength of the light that induces the binding of the first photo-specific binding protein and the second photo-specific binding protein depends on the kind of the first photo-specific binding protein and the second photo-specific binding protein, The wavelength of the light can be selected. That is, when CRY2 and CIBN are combined, light having a wavelength of 460 to 490 nm is irradiated, and when the light is not irradiated for 10 minutes or more, CRY2 and CIBN are dissociated from each other; When PhyB and PIF are combined, light having a wavelength of 650 nm is irradiated for 10 minutes, and light having a wavelength of 750 nm is irradiated for 5 minutes, PhyB and PIF dissociate from each other; When combining FKF1 and GIGANTEA, light with a wavelength of 460 nm can be irradiated for 30 minutes. In the embodiment of the present invention, light having a wavelength of 460 to 490 nm was irradiated to induce the binding of CIBN and CRY2.

According to the embodiment of the present invention, the expression of the fusion protein of CRY2 and mCherry and the fusion protein of CIB and CD9 was expressed in HEK293T cell, an immortal cell line producing a large amount of exosome. As a result, the distribution of mCherry protein uniformly distributed in cytoplasm When irradiated with blue light, it was observed that it is in a membrane such as a cell membrane and an endosome-like structure (Fig. 7). Similar results were also observed when FKF1 and mCherry fusion proteins and GIGANTEA and CD9 fusion proteins were expressed in HEK293T cells (Fig. 12). Furthermore, when the fusion proteins of CRY2 and mCherry and the fusion proteins of CIBN and CD9 were expressed in HEK293T and the intensity of blue light was controlled, it was found that mCherry protein captured in the exosomes (FIG. 9). As a result of treatment of HT1080 cells, which are isolated from exocytes produced from the cells, at a concentration of about 250 μg / ml, they did not show any cytotoxicity against HT1080 cells, MCherry protein was transferred to the cytoplasm of HT1080 cells (Fig. 10).

In order to compare the introduction efficiency of the target protein in the exosome of the present invention and the exosome transfer efficiency to the target cell with the conventional method, XPACK vector was used as the conventional method, and the CRY2 and mCherry proteins of the present invention Type fusion protein with CIBN and CD9 protein was introduced into HEK293T, and the amount of target protein produced in the exosome was compared. As a result, when the method of the present invention was used, (Fig. 15). In addition, when the exosome isolated from the exosome-producing cell is treated with target cells (HeLa) and the degree of expression of the target protein is compared, when the exosome isolated by the method of the present invention is used, And the highest expression was confirmed (Fig. 16).

The present invention provides, in another aspect, (a) a first expression vector comprising a polynucleotide encoding a fusion protein (first fusion protein) in which a first photo-specific binding protein and an exosome-specific marker are bound; And (b) a polynucleotide encoding a multicloning site into which a polynucleotide encoding a target protein can be introduced and a second photo-specific binding protein capable of binding to the first photo-specific binding protein, And a second expression vector comprising the exogenous expression vector.

In the vector for producing an exosome provided by the present invention, the exosome-specific marker, the first photo-specific binding protein, the exosome-producing cell and the second photo-specific binding protein are the same as described above.

The term "transforming cell for producing exosome" of the present invention refers to a cell that encodes a fusion protein (first fusion protein) in which exosome-specific markers and a first photo-specific binding protein are bound to the exosome- Refers to an exosome-producing cell into which a nucleotide has been introduced to express the first fusion protein.

In the present invention, the second expression vector may include a polynucleotide encoding the second photo-specific binding protein, and may be constructed to include a polyclonal region adjacent to the polynucleotide. The second expression vector When a polynucleotide encoding a target protein is introduced into the polyclonal region, the second light specific binding protein and the target protein can be expressed as a fusion form (second fusion protein).

The vector for producing exosome provided by the present invention can be used not only for transfecting cells and expression vectors for production of exosome but also for introducing the expression vector, culturing transgenic cells for production of exosome, producing transgenic cells for producing exosome One or more other component compositions, solutions or devices suitable for isolating and purifying exosomes may also be included. For example, it may further comprise an appropriate buffer necessary for introduction of the expression vector, a medium and a container necessary for culturing the transformed cell for producing the exosome, and the like.

In another aspect, the present invention provides an exosome prepared by the above method, wherein the Cas9 protein is contained therein.

The exosome produced by the above-mentioned method contains a fusion protein (first fusion protein) in which the exosome-specific marker and the first photo-specific binding protein are bound to the plasma membrane constituting the exosome, The second photo-specific binding protein capable of binding to the first photo-specific binding protein and the fusion protein (second fusion protein) in which the Cas9 protein is bound are contained in the cell, Through the fusion of the plasma membrane, the second fusion protein contained in the exosome can be transferred to the cytoplasm in the target tissue.

In addition, the present invention also provides an exosome carrying a Cas9 protein prepared by the above method and a DNA sequence engineering composition comprising a guide RNA (gRNA) specific to a target DNA sequence.

It is preferred, but not limited, that the composition induces a mutation in the normal sequence or corrects the mutation.

Mutations or mutations may be naturally occurring mutations or mutations. Mutations or mutations can be induced by pathogenic microorganisms. In other words, when a pathogenic microorganism is detected and the biological sample proved to be infected, the mutation or mutation is caused by an infection of the pathogenic microorganism. The pathogenic microorganism may be, but is not limited to, a virus or a bacteria.

As used herein, the term "guide RNA" refers to an RNA specific for a target DNA, which can form a complex with the Cas protein and bring the Cas protein to the target DNA.

In the present invention, the guide RNA may be composed of two RNAs, i.e., CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA), or may be composed of a single And may be single-chain RNA (sgRNA).

The guide RNA may be a dual RNA including crRNA and tracrRNA. Any guide RNA may be used in the present invention if the guide RNA comprises a portion complementary to an essential portion and target of the crRNA and the tracrRNA. The crRNA may be hybridized with the target DNA.

The guide RNA may further comprise one or more additional nucleotides at the 5 ' end of the single-chain guide RNA or the crRNA of the double RNA.

Preferably, the guide RNA may further comprise two additional guanine nucleotides at the 5 'end of the single-chain guide RNA or the double-stranded RNA's crRNA. The guide RNA can be delivered to the cell or organism in the form of RNA or in the form of DNA encoding the guide RNA. The guide RNA may be in the form of isolated RNA, RNA contained in the viral vector, or in a form encoded in a vector. Preferably, the vector may be a viral vector, a plasmid vector, or an agrobacterium vector, but is not limited thereto.

The DNA encoding the guide RNA may be a vector containing a sequence encoding the guide RNA. For example, a plasmid DNA comprising a promoter and a sequence encoding a separate guide RNA or guide RNA may be transfected into a cell or an organism to deliver the guide RNA to the cell or organism. Alternatively, the guide RNA can be delivered to cells or organisms using virus-mediated gene transfer.

When the guide RNA is transfected into a cell or organism in the form of isolated RNA, the guide RNA may be produced by in vitro transcription using any in vitro transcription system known in the art. The guide RNA is preferably delivered to the cell in the form of RNA separated from the form of the plasmid comprising the sequence encoding the guide RNA. As used herein, the term "isolated RNA" can be used interchangeably with "naked RNA ". This saves cost and time because it does not require a cloning step. However, the use of plasmid DNA or virus-mediated gene transfer for the transfection of guide RNA is not excluded.

In a specific embodiment of the present invention, the present inventors used DNA encoding pCAG-loxP-STOP-loxP-ZsGreen to identify the function of Cas9 recombinase using exosome (Cas9: EXPLOR) HeLa cells were transfected with Cas9: EXPLOR or Negative: EXPLOR (Exosome without Cas9 recombinase), or pCMV-Cas9 vector was transfected to measure the expression of green fluorescent ZsGreen reporter protein. Results: In the Cas9: EXPLOR treated HT1080 cells and HeLa cells, ZsGreen expression was confirmed, which showed the same trend as the result of transformation with the pCMV-Cas9 vector as a positive control (see FIG. 19).

Accordingly, the exosome (Cas9: EXPLOR) carrying the Cas9 protein of the present invention can be used as a composition for genetic correction, and thus can be used for delivery technology of a genome editing system.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples.

However, the following Examples and Experimental Examples are intended to illustrate the present invention, and the scope of the present invention is not limited to these Examples and Experimental Examples.

Exosome  Produce

<1-1> Confirmation of binding of CIBN and CRY2 for the production of exosome

The pcDNA3.1 (+) vector containing the CIBN-EGFP-CD9 gene and the pcDNA3.1 (+) vector containing the mCherry-CRY2 gene were introduced into the exosome-producing HEK293T cells under light-free conditions, , Then replaced with medium without fetal bovine serum, and further cultured for 48 hours. After completion of the cultivation, blue light having a wavelength of 460 to 490 nm was irradiated, and the positions of red fluorescence appearing in the mCherry before and after irradiation of the blue light were confirmed through a confocal microscope (Fig. 7).

FIG. 7 is a fluorescence photograph showing the change in the intracellular location of mCherry protein in a transformant into which CIBN-EGFP-CD9 gene and mCherry-CRY2 gene are introduced into HEK293T cells according to irradiation of blue light. As shown in FIG. 7, mCherry protein is uniformly distributed in the cytoplasm before blue light that induces the binding of CIBN and CRY2, which is a photo-specific binding protein, is observed. However, after the blue light is irradiated, Of the total population. This clustering of mCherry protein was analyzed to be caused by the combination of CIBN and CRY2, a light specific binding protein.

<1-2> Confirmation of binding of GIGANTEA and FKF1 for the production of exosome

Using pcDNA3.1 (+) vector containing GIGANTEA-EGFP-CD9 gene and pcDNA3.1 (+) vector containing mCherry-FKF1LOV gene and using the same method as in Example <1-1> Exosomes in the cells were confirmed. (In the above FKF1LOV, LOV is an abbreviation of light-oxygen-voltage domain. It indicates a domain in which FKF1 protein actually binds to another protein by light. Thus, FKF1 and FKF1LOV are used in the same meaning.)

As shown in FIG. 12, the mCherry protein is uniformly distributed in the cytoplasm before the blue light that induces the binding of GIGANTEA and FKF1, which is a photo-specific binding protein, is irradiated. However, The mCherry protein was dense in the membrane (Fig. 12). Thus, similar to the results of <1-1>, it was confirmed that the dense mCherry protein can be induced by the binding of GIGANTEA and FKF1 of the photo-specific binding protein.

Exosome  Production and Exosome  Effect of light intensity on production

Each of the expression vectors containing the CIBN-EGFP-CD9 gene and the mCherry-CRY2 gene, respectively, was irradiated with light of a wavelength of 460 nm at an intensity of 0, 5, 20, 50 or 200 μW, , HEK293T cells, cultured for 24 hours, then replaced with medium without fetal bovine serum, and further cultured for 48 hours. After completion of the culture, the culture broth was separated and centrifuged (3000 × g, 15 minutes) to obtain a supernatant from which cellular debris was removed. The obtained supernatant was mixed with ExoQuick-TC Exosome Precipitation Solution (System Biosciences, Mountain View, California, USA) of 5 times the volume of the supernatant and centrifuged (1500 x g, 30 minutes) Cotton was obtained, and the resulting exosome was suspended in PBS to give an exosome suspension. The exosome suspension was filtered through a 0.2 탆 filter using a syringe equipped with a 27-G needle to obtain a single-sized exosome (Fig. 8). Then, Exosome Lysate was prepared using Lysis buffer, and the amount of mCherry protein contained in exosome was compared through immunoblot analysis (FIG. 9).

FIG. 9 is an immunoblot analysis image showing the result of measurement of a change in the content of a target protein (mCherry protein) captured inside the exosome according to the intensity of blue light. As shown in FIG. 9, when the blue light was irradiated at an intensity of 20 to 50 μW, it was confirmed that the content of the target protein (mCherry protein) captured in the exosome exhibited the maximum value. From the above results, it was found that the content of the target protein captured in the exosome can be controlled by controlling the intensity of the light irradiated upon binding of the photo-specific binding protein.

Exosome  Treatment effect

Each of the expression vectors containing the CIBN-EGFP-CD9 gene and the mCherry-CRY2 gene was introduced into HEK293T cells, which are exosome-producing cells, under the LEDs that emit light with a wavelength of 460 nm at an intensity of 50 μW Exosome was extracted by the method of Example 2. Then, the extracted exosome was treated with HT1080 cells at a concentration of 250 μg / ml for 24 hours. Then, the HT1080 cells were fixed with 0.1 M phosphate buffer (pH 7.4) containing 4% PFA and 0.01% GA and fixed on 10% gelatin gel. The cells adhered onto the gelatin gel were cooled with liquid nitrogen for one day and a slice cut to a thickness of 45 nm was obtained at -120 캜 using a cryoultramicrotome. Next, the flakes were immunostained with anti-mCherry antibody and protein A-gold, and mCherry protein was observed through Tecnai G2 Spirit Twin TEM (FIG. 10).

FIG. 10 is an electron micrograph showing the result of treating target cells (HT1080 cells) treated with an exosome containing a target protein (mCherry) and confirming the introduction of a target protein into the target cell. And the right side represents the target cell treated with exosome. As shown in FIG. 10, when the exosome of the present invention was treated on the target cell, it was confirmed that the target protein was transferred into the target cell.

Containing the desired protein Exosome  analysis

Each of the expression vectors containing the CIBN-EGFP-CD9 gene and the mCherry-CRY2 gene was introduced into HEK293T cells, which are exosome-producing cells, under the LEDs that emit light with a wavelength of 460 nm at an intensity of 50 μW Exosome was extracted by the method of Example 2. Then, the extracted exosome was treated with HT1080 cells at a concentration of 250 μg / ml for 24 hours. Then, the red fluorescence of mCherry protein was confirmed by fluorescence microscopy, and the ratio of dead cells of cells treated with exosome and untreated cells was compared through LDH cell death assay (FIG. 11).

FIG. 11 is a graph showing fluorescence (a) showing the result of examining whether the target protein is introduced into the target cell (HT1080 cell) after treating the exosome containing the target protein (mCherry) to the target cell (B) showing the results obtained by comparing the ratios of the apoptotic cells induced by the apoptotic cells. As shown in Fig. 11, it was confirmed that apoptosis did not induce apoptosis.

Exosome  Produced and produced Exosome  Comparison of introduction efficiency of target protein

<5-1> Confirmation of exosome production efficiency

In order to confirm the expression of the target protein in the exosome-producing cells in order to compare the exosome production of the present invention and the degree of introduction of the target protein in the produced exosome, the luciferase activity measurement experiment was performed .

In the conventional method, XPACK-Luciferase-mCherry was introduced into HEK293T cells (XP) using XPACK (Systems Biosciences), which is a commercial vector designed for exosome mounting technology, and Luciferase-mCherry-CRY2 And CIBN-EGFP-CD9 were introduced into HEK293T cells (EXPLOR), and then the intracellular luciferase activity was measured to compare the efficiency of the two methods. The luciferase activity was measured according to the manufacturer's instructions (Luciferase Assay Reagent, Promega), and the standard curve of the result was plotted, and then the number of exocytes in the cell was quantitatively calculated.

As shown in FIG. 14, it was confirmed that the method using the photo-specific binding proteins CIBN and CRY2 of the present invention was significantly higher in the introduction efficiency into exosomes than the conventional method XP (FIG. 14).

<5-2> Expression of the target protein in the produced exosome

The cells of the above Example <5-1> were cultured for 72 hours, and the exosomes were separated (Exoquick-TC, Systems biosciences) As a result of indirectly comparing the amount of the target protein with the activity of luciferase activity, as shown in FIG. 15, the method of the present invention can produce an exosome containing a remarkably large amount of the target protein (Fig. 15).

<5-3> Comparison of introduction efficiency of target protein

Using the measured luciferase activity values in the above Examples <5-1> and <5-2>, the introduction efficiency (E) of the target protein was calculated by the following equation (1).

[Equation 1]

As shown in FIG. 15, it was confirmed that the exosomes produced using the CRY2 and CIBN binding of the present invention were 4 to 120 times more efficient than the other comparative groups (FIG. 15).

Target cell Exosome  Comparison of transfer efficiency

In order to compare the efficiency of treatment of the exosome into which the target protein was introduced to the target cells, 5 × 10 ⁹ exosomes were treated with HeLa cells for 24 hours and the fluorescence intensity expressed in the cells was measured. As a result, As shown, it was confirmed that the fluorescence intensity of EXPLOR of the present invention was remarkably high (FIG. 16).

Therefore, it can be seen that the method of using the exosome of the present invention can more effectively deliver the target protein to the target cell than the conventional method.

< Experimental Example  1 > cas9 protein Of exosomes (Cas9: EXPLOR)  Produce

<1-1> Exosome  Identification of internal Cas9 protein

In order to confirm that the Cas9 protein having the amino acid sequence of SEQ ID NO: 1 is loaded on the exosome, the present inventors investigated whether binding of CIBN and CRY2 in cells expressing CIBN-EGFP-CD9 and Cas9-mCherry-CRY2 gene Respectively.

As shown in FIG. 19, the pcDNA3.1 (+) vector into which the Cas9-mCherry-CRY2 gene was inserted had a total length of 11,890 base pairs, and Cas9 , mCherry and Cryptochrome 2 are linked by a linker sequence of 45 base pairs and 27 base pairs in length, respectively. Each part protein has a length of 4194 base pairs, 699 base pairs, and 1497 base pairs.

Specifically, the pcDNA3.1 (+) vector containing the CIBN-EGFP-CD9 gene and the pcDNA3.1 (+) vector containing the Cas9-mCherry-CRY2 gene were transfected into the exosome-producing HEK293T cells , Cultured for 24 hours, then replaced with a medium containing no fetal bovine serum, and further cultured for 48 hours. After completion of the cultivation, blue light having a wavelength of 488 nm was irradiated, and the positions of red fluorescence appearing in the mCherry before and after irradiation of the blue light were confirmed through a confocal microscope. The experiment was repeated 5 or more times.

As a result, Cas9-mCherry-CRY2 (red) was found to bind CIBN-EGFP-CD9 by blue light stimulation as shown in Fig. 18 (Fig. 18). Therefore, it was confirmed that Cas9 protein was loaded inside the exosome.

&Lt; 2-2 > Of exosomes (Cas9: EXPLOR)  production

The present inventors conducted the following experiment to obtain an exosome carrying Cas9 protein.

Specifically, each expression vector containing CIBN-EGFP-CD9 gene and Cas9-mCherry-CRY2 gene was introduced into HEK293T cells, which are exosome-producing cells, and cultured for 24 hours. Then, , And further cultured for 48 hours under an LED emitting light of 488 nm at an intensity of 50 μW. After the incubation was completed, the culture broth was separated and centrifuged (2000 xg, 15 minutes) to obtain a supernatant from which cellular debris was removed. To the supernatant obtained above, 5 times volume of the supernatant was mixed with ExoQuick-TC Exosome Precipitation Solution (System Biosciences, Mountain View, California, USA) at 4 ° C for 18 hours, centrifuged (1500 × g, 30 minutes) to obtain a precipitated exosome. The resulting exosome was suspended in PBS to obtain an exosome suspension (FIG. 8).

In addition, for the mass production of exosomes, HEK293T cells, which are exosome-producing cells stably expressing the respective expression vectors including the CIBN-EGFP-CD9 gene and the Cas9-mCherry-CRY2 gene, And cultured for 48 to 72 hours under an atmosphere of light of 508 W at 488 nm. After the incubation was completed, the culture broth was separated and centrifuged (2000 xg, 15 minutes) to obtain a supernatant from which cellular debris was removed. The supernatant was filtered with 0.2 ul PES membrane (Corning) to remove particles greater than 200 nm. Tangential Flow Filtration (TFF) filtration was used to remove particles less than 20 nm from the same filtrate and to concentrate and purify the exosome from the filtrate. The membrane of TFF was a Vivaflow 50-100 KDa PES membrane (Sartorius). The filtrate was repeatedly rotated at a TFF air pressure of 1.5 to 2 to concentrate and purify the exosome. Thereafter, the exocom-concentration was transferred to an Amicon Ultra-0.5 (100 kDa) (Millipore) filter, centrifuged at 10,000 to 14,000 g for 5 minutes to remove the aqueous solution, and then the buffer was filled in a buffer suitable for the purpose of the experiment. And the final exosome was obtained.

<110> Korea Advanced Institute of Science and Technology          Cellex Life Sciences Inc. <120> Technology to deliver the genome editing tool by using the          CRISPR-CAS family <130> 2016P-07-013 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 1409 <212> PRT <213> Homo sapiens <400> 1 Met Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr Ala   1 5 10 15 Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala Ala Asp              20 25 30 Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val Gly Trp          35 40 45 Ala Val Ile Thr Asp Glu Tyr Lys Val Ser Lys Lys Phe Lys Val      50 55 60 Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala  65 70 75 80 Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg                  85 90 95 Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu             100 105 110 Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe         115 120 125 His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu     130 135 140 Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu 145 150 155 160 Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr                 165 170 175 Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile             180 185 190 Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn         195 200 205 Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln     210 215 220 Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala 225 230 235 240 Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile                 245 250 255 Ala Gln Leu Pro Gly Gly Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile             260 265 270 Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu         275 280 285 Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp     290 295 300 Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe 305 310 315 320 Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu                 325 330 335 Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile             340 345 350 Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu         355 360 365 Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln     370 375 380 Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu 385 390 395 400 Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr                 405 410 415 Glu Glu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln             420 425 430 Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu         435 440 445 Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys     450 455 460 Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr 465 470 475 480 Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr                 485 490 495 Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val             500 505 510 Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe         515 520 525 Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu     530 535 540 Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val 545 550 555 560 Thr Glu Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys                 565 570 575 Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys             580 585 590 Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val         595 600 605 Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr     610 615 620 His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu 625 630 635 640 Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe                 645 650 655 Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu             660 665 670 Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly         675 680 685 Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln     690 695 700 Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn 705 710 715 720 Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu                 725 730 735 Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His Glu             740 745 750 His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu         755 760 765 Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg His     770 775 780 Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr 785 790 795 800 Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu Glu                 805 810 815 Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val Glu             820 825 830 Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn         835 840 845 Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser     850 855 860 Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp 865 870 875 880 Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys                 885 890 895 Ser Asp Asn Val Ser Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr             900 905 910 Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp         915 920 925 Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala     930 935 940 Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His 945 950 955 960 Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn                 965 970 975 Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu             980 985 990 Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile         995 1000 1005 Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly    1010 1015 1020 Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr 1025 1030 1035 1040 Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser Glu                1045 1050 1055 Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile            1060 1065 1070 Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg        1075 1080 1085 Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp    1090 1095 1100 Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro 1105 1110 1115 1120 Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser                1125 1130 1135 Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg            1140 1145 1150 Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr        1155 1160 1165 Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser    1170 1175 1180 Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu 1185 1190 1195 1200 Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly                1205 1210 1215 Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser            1220 1225 1230 Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly        1235 1240 1245 Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn    1250 1255 1260 Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu 1265 1270 1275 1280 Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr Leu                1285 1290 1295 Asp Glu Ile Ile Glu Glu Ile Ser Glu Phe Ser Lys Arg Val Ile Leu            1300 1305 1310 Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg        1315 1320 1325 Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr    1330 1335 1340 Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr 1345 1350 1355 1360 Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr                1365 1370 1375 Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu            1380 1385 1390 Ser Gln Leu Gly Gly Asp Ser Arg Ala Asp Pro Lys Lys Lys Arg Lys        1395 1400 1405 Val     <210> 2 <211> 1300 <212> PRT <213> Homo sapiens <400> 2 Met Ser Ile Tyr Gln Glu Phe Val Asn Lys Tyr Ser Leu Ser Lys Thr   1 5 10 15 Leu Arg Phe Glu Leu Ile Pro Gln Gly Lys Thr Leu Glu Asn Ile Lys              20 25 30 Ala Arg Gly Leu Ile Leu Asp Asp Glu Lys Arg Ala Lys Asp Tyr Lys          35 40 45 Lys Ala Lys Gln Ile Ile Asp Lys Tyr His Gln Phe Phe Ile Glu Glu      50 55 60 Ile Leu Ser Ser Val Cys Ile Ser Glu Asp Leu Leu Gln Asn Tyr Ser  65 70 75 80 Asp Val Tyr Phe Lys Leu Lys Lys Ser Asp Asp Asp Asn Leu Gln Lys                  85 90 95 Asp Phe Lys Ser Ala Lys Asp Thr Ile Lys Lys Gln Ile Ser Glu Tyr             100 105 110 Ile Lys Asp Ser Glu Lys Phe Lys Asn Leu Phe Asn Gln Asn Leu Ile         115 120 125 Asp Ala Lys Lys Gly Gln Glu Ser Asp Leu Ile Leu Trp Leu Lys Gln     130 135 140 Ser Lys Asp Asn Gly Ile Glu Leu Phe Lys Ala Asn Ser Asp Ile Thr 145 150 155 160 Asp Ile Asp Glu Ala Leu Glu Ile Ile Lys Ser Phe Lys Gly Trp Thr                 165 170 175 Thr Tyr Phe Lys Gly Phe His Glu Asn Arg Lys Asn Val Tyr Ser Ser             180 185 190 Asn Asp Ile Pro Thr Ser Ile Ile Tyr Arg Ile Val Asp Asp Asn Leu         195 200 205 Pro Lys Phe Leu Glu Asn Lys Ala Lys Tyr Glu Ser Leu Lys Asp Lys     210 215 220 Ala Pro Glu Ala Ile Asn Tyr Glu Gln Ile Lys Lys Asp Leu Ala Glu 225 230 235 240 Glu Leu Thr Phe Asp Ile Asp Tyr Lys Thr Ser Glu Val Asn Gln Arg                 245 250 255 Val Phe Ser Leu Asp Glu Val Phe Glu Ile Ala Asn Phe Asn Asn Tyr             260 265 270 Leu Asn Gln Ser Gly Ile Thr Lys Phe Asn Thr Ile Ile Gly Gly Lys         275 280 285 Phe Val Asn Gly Glu Asn Thr Lys Arg Lys Gly Ile Asn Glu Tyr Ile     290 295 300 Asn Leu Tyr Ser Gln Gln Ile Asn Asp Lys Thr Leu Lys Lys Tyr Lys 305 310 315 320 Met Ser Val Leu Phe Lys Gln Ile Leu Ser Asp Thr Glu Ser Ser Ser Ser                 325 330 335 Phe Val Ile Asp Lys Leu Glu Asp Asp Ser Asp Val Val Thr Thr Met             340 345 350 Gln Ser Phe Tyr Glu Gln Ile Ala Ala Phe Lys Thr Val Glu Glu Lys         355 360 365 Ser Ile Lys Glu Thr Leu Ser Leu Leu Phe Asp Asp Leu Lys Ala Gln     370 375 380 Lys Leu Asp Leu Ser Lys Ile Tyr Phe Lys Asn Asp Lys Ser Leu Thr 385 390 395 400 Asp Leu Ser Gln Gln Val Phe Asp Asp Tyr Ser Val Ile Gly Thr Ala                 405 410 415 Val Leu Glu Tyr Ile Thr Gln Gln Ile Ala Pro Lys Asn Leu Asp Asn             420 425 430 Pro Ser Lys Lys Glu Glu Glu Leu Ile Ala Lys Lys Thr Glu Lys Ala         435 440 445 Lys Tyr Leu Ser Leu Glu Thr Ile Lys Leu Ala Leu Glu Glu Phe Asn     450 455 460 Lys His Arg Asp Ile Asp Lys Gln Cys Arg Phe Glu Glu Ile Leu Ala 465 470 475 480 Asn Phe Ala Ala Ile Pro Met Ile Phe Asp Glu Ile Ala Gln Asn Lys                 485 490 495 Asp Asn Leu Ala Gln Ile Ser Ile Lys Tyr Gln Asn Gln Gly Lys Lys             500 505 510 Asp Leu Leu Gln Ala Ser Ala Glu Asp Asp Val Lys Ala Ile Lys Asp         515 520 525 Leu Leu Asp Gln Thr Asn Asn Leu Leu His Lys Leu Lys Ile Phe His     530 535 540 Ile Ser Gln Ser Glu Asp Lys Ala Asn Ile Leu Asp Lys Asp Glu His 545 550 555 560 Phe Tyr Leu Val Phe Glu Glu Cys Tyr Phe Glu Leu Ala Asn Ile Val                 565 570 575 Pro Leu Tyr Asn Lys Ile Arg Asn Tyr Ile Thr Gln Lys Pro Tyr Ser             580 585 590 Asp Glu Lys Phe Lys Leu Asn Phe Glu Asn Ser Thr Leu Ala Asn Gly         595 600 605 Trp Asp Lys Asn Lys Glu Pro Asp Asn Thr Ala Ile Leu Phe Ile Lys     610 615 620 Asp Asp Lys Tyr Tyr Leu Gly Val Met Asn Lys Lys Asn Asn Lys Ile 625 630 635 640 Phe Asp Asp Lys Ala Ile Lys Glu Asn Lys Gly Glu Gly Tyr Lys Lys                 645 650 655 Ile Val Tyr Lys Leu Leu Pro Gly Ala Asn Lys Met Leu Pro Lys Val             660 665 670 Phe Phe Ser Ala Lys Ser Ile Lys Phe Tyr Asn Pro Ser Glu Asp Ile         675 680 685 Leu Arg Ile Arg Asn His Ser Thr His Thr Lys Asn Gly Ser Pro Gln     690 695 700 Lys Gly Tyr Glu Lys Phe Glu Phe Asn Ile Glu Asp Cys Arg Lys Phe 705 710 715 720 Ile Asp Phe Tyr Lys Gln Ser Ile Ser Lys His Pro Glu Trp Lys Asp                 725 730 735 Phe Gly Phe Arg Phe Ser Asp Thr Gln Arg Tyr Asn Ser Ile Asp Glu             740 745 750 Phe Tyr Arg Glu Val Glu Asn Gln Gly Tyr Lys Leu Thr Phe Glu Asn         755 760 765 Ile Ser Glu Ser Tyr Ile Asp Ser Val Val Asn Gln Gly Lys Leu Tyr     770 775 780 Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ser Ala Tyr Ser Lys Gly Arg 785 790 795 800 Pro Asn Leu His Thr Leu Tyr Trp Lys Ala Leu Phe Asp Glu Arg Asn                 805 810 815 Leu Gln Asp Val Val Tyr Lys Leu Asn Gly Glu Ala Glu Leu Phe Tyr             820 825 830 Arg Lys Gln Ser Ile Pro Lys Lys Ile Thr His Pro Ala Lys Glu Ala         835 840 845 Ile Ala Asn Lys Asn Lys Asp Asn Pro Lys Lys Glu Ser Val Phe Glu     850 855 860 Tyr Asp Leu Ile Lys Asp Lys Arg Phe Thr Glu Asp Lys Phe Phe Phe 865 870 875 880 His Cys Pro Ile Thr Ile Asn Phe Lys Ser Ser Gly Ala Asn Lys Phe                 885 890 895 Asn Asp Glu Ile Asn Leu Leu Leu Lys Glu Lys Ala Asn Asp Val His             900 905 910 Ile Leu Ser Ile Asp Arg Gly Glu Arg His Leu Ala Tyr Tyr Thr Leu         915 920 925 Val Asp Gly Lys Gly Asn Ile Ile Lys Gln Asp Thr Phe Asn Ile Ile     930 935 940 Gly Asn Asp Arg Met Lys Thr Asn Tyr His Asp Lys Leu Ala Ala Ile 945 950 955 960 Glu Lys Asp Arg Asp Ser Ala Arg Lys Asp Trp Lys Lys Ile Asn Asn                 965 970 975 Ile Lys Glu Met Lys Glu Gly Tyr Leu Ser Gln Val Val His Glu Ile             980 985 990 Ala Lys Leu Val Ile Glu Tyr Asn Ale Ile Val Val Phe Glu Asp Leu         995 1000 1005 Asn Phe Gly Phe Lys Arg Gly Arg Phe Lys Val Glu Lys Gln Val Tyr    1010 1015 1020 Gln Lys Leu Glu Lys Met Leu Ile Glu Lys Leu Asn Tyr Leu Val Phe 1025 1030 1035 1040 Lys Asp Asn Glu Phe Asp Lys Thr Gly Gly Val Leu Arg Ala Tyr Gln                1045 1050 1055 Leu Thr Ala Pro Phe Glu Thr Phe Lys Lys Met Gly Lys Gln Thr Gly            1060 1065 1070 Ile Ile Tyr Tyr Val Pro Ala Gly Phe Thr Ser Lys Ile Cys Pro Val        1075 1080 1085 Thr Gly Phe Val Asn Gln Leu Tyr Pro Lys Tyr Glu Ser Val Ser Lys    1090 1095 1100 Ser Gln Glu Phe Phe Ser Lys Phe Asp Lys Ile Cys Tyr Asn Leu Asp 1105 1110 1115 1120 Lys Gly Tyr Phe Glu Phe Ser Phe Asp Tyr Lys Asn Phe Gly Asp Lys                1125 1130 1135 Ala Ala Lys Gly Lys Trp Thr Ile Ala Ser Phe Gly Ser Arg Leu Ile            1140 1145 1150 Asn Phe Arg Asn Ser Asp Lys Asn His Asn Trp Asp Thr Arg Glu Val        1155 1160 1165 Tyr Pro Thr Lys Glu Leu Glu Lys Leu Leu Lys Asp Tyr Ser Ile Glu    1170 1175 1180 Tyr Gly His Gly Glu Cys Ile Lys Ala Ala Ile Cys Gly Glu Ser Asp 1185 1190 1195 1200 Lys Lys Phe Phe Ala Lys Leu Thr Ser Val Leu Asn Thr Ile Leu Gln                1205 1210 1215 Met Arg Asn Ser Lys Thr Gly Thr Glu Leu Asp Tyr Leu Ile Ser Pro            1220 1225 1230 Val Ala Asp Val Asn Gly Asn Phe Phe Asp Ser Arg Gln Ala Pro Lys        1235 1240 1245 Asn Met Pro Gln Asp Ala Asp Ala Asn Gly Ala Tyr His Ile Gly Leu    1250 1255 1260 Lys Gly Leu Met Leu Leu Gly Arg Ile Lys Asn Asn Gln Glu Gly Lys 1265 1270 1275 1280 Lys Leu Asn Leu Val Ile Lys Asn Glu Glu Tyr Phe Glu Phe Val Gln                1285 1290 1295 Asn Arg Asn Asn            1300

Claims (16)

A method for mass-producing an exosome comprising Cas9 or Cpf1 protein, comprising the steps of:
a) a polynucleotide encoding a fusion protein (first fusion protein) in which the exosome-specific marker and the first photo-specific binding protein are bound to the exosome-producing cell, and a polynucleotide that binds to the first photo- Introducing a polynucleotide encoding a fusion protein (second fusion protein) in a form in which Cas9 or Cpf1 protein is bound to a second photo-specific binding protein capable of binding to a second fusion protein;
b) irradiating the exosome-producing cell with light capable of inducing binding of the first photo-specific binding protein and the second photo-specific binding protein; And
c) after the exosome is produced in the exosome-producing cell, stopping the irradiation of the light.
delete The method according to claim 1, wherein the Cas9 protein is composed of the amino acid sequence of SEQ ID NO: 1 and the Cpf1 protein is composed of the amino acid sequence of SEQ ID NO: 2. A method for mass production.
The method of claim 1, wherein the exosome-producing cell is one or more cells selected from the group consisting of B-lymphocytes, T-lymphocytes, dendritic cells, megakaryocytes, macrophages, stem cells and tumor cells A method for producing a large amount of exosome comprising the Cas9 or Cpf1 protein.
The method according to claim 1, wherein the exosome-specific marker is CD9, CD63, CD81 or CD82.
delete 2. The method of claim 1, wherein the first photo-specific binding protein is selected from the group consisting of cryptochrome-interacting basichelix-loop-helix protein (CIBN), N-terminal domain of CIB, phytochrome B, phytochrome interacting factor (PIF) , FKF1 (Flavinbinding, Kelch repeat, Fbox 1), GIGANTEA, CRY (chryptochrome) and PHR (phytolyase homologous region) A method for mass production.
The method of claim 1, wherein when the first photo-specific binding protein is CIB or CIBN, the second photo-specific binding protein is CRY or PHR, and the second photo- Wherein the binding of the protein is carried out by irradiating light having a wavelength of 460 to 490 nm, wherein the Cas9 or Cpf1 protein is produced in a large amount.
The method of claim 1, wherein when the first photo-specific binding protein is PhyB, the second photo-specific binding protein is PIF, and the binding of the first photo- specific binding protein and the second photo- Wherein the method comprises the step of irradiating light having a wavelength of 600 to 650 nm to produce a large amount of exosome comprising Cas9 or Cpf1 protein.
The method of claim 1, wherein when the first photo-specific binding protein is GIGANTEA, the second photo-specific binding protein is FKF1, and the binding of the first photo- specific binding protein and the second photo- Wherein the method comprises irradiating light having a wavelength ranging from 460 to 490 nm to produce a large amount of exosome comprising Cas9 or Cpf1 protein.
An exosome comprising Cas9 or Cpf1 protein incorporated therein, prepared using the method of claim 1. &lt; Desc / Clms Page number 36 &gt;
A method for delivering Cas9 or Cpf1 protein to the cytoplasm in vitro in an exocytosis using the exosome of claim 11.
A DNA sequence manipulating composition comprising the exosome of claim 11 and a guide RNA (gRNA) specific to the target DNA sequence.
14. The composition of claim 13, wherein said composition induces mutation in normal sequence or corrects mutation.
A composition for preparing exosome, comprising:
(a) a first expression vector comprising a polynucleotide encoding a fusion protein (first fusion protein) in which a first light-specific binding protein and an exosome-specific marker are bound; And
(b) a polynucleotide capable of introducing a polynucleotide encoding Cas9 or Cpf1 protein and a polynucleotide encoding a second photo-specific binding protein capable of binding to the first photo-specific binding protein 2 expression vector.
[16] The method of claim 15, wherein the second expression vector is a fusion protein (second fusion protein) in which the second photo-specific binding protein is fused with Cas9 or Cpf1 protein in an exosome-producing cell, &Lt; / RTI &gt;

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