Development of CRISPR-Cas9 vector for genome editing in animal cells

Abstract

The present invention relates to a CRISPR Cas9 vector and its uses, and more specifically, to a CRISPR Cas9 vector with increased gene editing efficiency. The CRISPR Cas9 vector of the present invention expresses the RAD51 gene, and when the CRISPR Cas9 vector of the present invention is used, the efficiency of gene editing increases by more than 2 times, so genes can be edited with higher efficiency and accuracy.

Description

Technology Field

[0001] The present invention relates to a CRISPR-Cas9 vector and its uses, and more specifically, to a CRISPR Cas9 vector with increased gene editing efficiency. Background Technology

[0003] The Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR) / CRISPR-Associated Protein 9 (Cas9) system acts as a secondary defense immune system in bacteria and archaea. When a virus infects bacteria, this system integrates fragments of the virus's DNA into the bacterial genome. Subsequently, during a secondary invasion, the viral DNA sequence present in the bacteria is transcribed, and the transcript causes a nuclease to move to a DNA sequence complementary to itself, thereby destroying the genome of the invading virus.

[0004] The CRISPR / Cas9 system has been developed as a gene editing technology in eukaryotes, requiring three essential components: the Cas9 protein, sequence recognition RNA, and trans-activating crisper RNA. In this context, the sequence recognition RNA and trans-activating crisper RNA are synthesized into a single RNA molecule and are being newly used in gene editing technology as a single guide RNA (sgRNA). The sequence recognition RNA and trans-activating crisper RNA are collectively referred to as gRNA, and the structure of gRNA significantly influences the Cas9 activation efficiency. Through various studies, it has been concluded that acting as a combined sgRNA exhibits higher Cas9 activation efficiency than using the two types of gRNA separately.

[0005] sgRNA binds complementarily to the target DNA sequence and simultaneously binds to Cas9, enabling Cas9 to function on the target DNA. As a nuclease, the Cas9 protein is moved to the target sequence by sgRNA; thanks to the PAM (Protospacer adjacent motif) sequence present on the genome, it is able to bind to the genome and form a DNA double-strand break in a specific region. In response, a DNA double-strand damage repair process occurs, and due to the sequence altered during this repair, normal expression of the corresponding gene becomes difficult, resulting in the gene failing to perform its proper function.

[0006] When double-strand damage occurs on DNA due to nucleases, a repair process takes place. These repair processes include non-homologous end joining (NHEJ), which involves deleting a portion of the damaged site and simply connecting the damaged DNA ends, and homology-directed repair (HDR), which involves obtaining homologous sequences for the damaged site from homologous or sister chromatids and performing repair based on them.

[0007] In this case, RAD51 is responsible for the function of enabling homology search to obtain repair information regarding the damaged site from homologous chromatids or sister chromatids during the repair process through homologous recombination that occurs after double-strand damage, thereby allowing appropriate recombination to take place at the damaged site.

[0008] Targeted genome editing is a technology that enables research into the function and characteristics of specific genes by utilizing nucleases that recognize and act on specific regions of the genome. These targeted genome editing technologies include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the aforementioned Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR) / CRISPR-Associated Protein 9 (Cas9) systems. Among the previously introduced targeted genome editing technologies, ZFNs and TALENs are not only complex but also costly; consequently, the CRISPR-Cas9 system is generally preferred. Furthermore, because it can be applied to a wide variety of cell and animal models without limitations, it serves as a simple and economical method for the treatment and analysis of diseases. Despite this popularity, the CRISPR-Cas9 system still has limitations.

[0009] The specificity of the Cas9 protein to its target is determined by the approximately 20-nt nucleotide sequence of the sgRNA and the presence or absence of PAM sequences on the genome. In this case, off-target editing can occur due to base mismatch binding, where binding takes place even if the complementarity between the sgRNA targeting sequence and the genomic target sequence is not perfect. Alternatively, the secondary structure of the sgRNA may interfere with the binding between the sgRNA and the target gene. Furthermore, the addition of epigenetic changes on the genome may also make sgRNA binding difficult. Also, unlike other types of enzymes, Cas9 forms a highly energy-stable binding with both the sgRNA and the protospacer DNA, preventing easy separation. From the perspective of the enzyme and substrate, once the enzyme-substrate complex binds, an enzyme-product complex is formed through the enzyme's function. In this context, the speed at which the enzyme detaches from the enzyme-product complex determines whether it can bind to other substrates and perform its function smoothly. However, due to Cas9's characteristic of forming energetically stable bonds with substrates, it cannot continuously and fully demonstrate its gene editing capabilities through reactions with other substrates. Consequently, there is currently a lack of technologies that reduce the frequency of off-target editing while ensuring that gene editing occurs only on appropriate targets, as well as technologies that enable the full functioning of Cas9. Prior art literature

[0011] 1) Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816-821. (2012).2) Gasiunas, Giedrius et al. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proceedings of the National Academy of Sciences of the United States of America vol. 109,39 (2012): E2579-86.3) Sander, J., Joung, J. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol 32, 347-355. The problem to be solved

[0012] The inventors have made diligent research efforts to improve the gene editing efficiency of the CRISPR / Cas9 system. As a result, they confirmed that inducing the overexpression of RAD51 together with the CRISPR / Cas9 system improves the efficiency of the target gene editing action of the CRISPR / Cas9 system, and thus completed the present invention.

[0013] Therefore, the objective of the present invention is to RAD51 The purpose is to provide a CRISPR / Cas9 vector containing a gene.

[0014] Another objective of the present invention is to provide a genome editing composition comprising the above vector.

[0015] Another objective of the present invention is to provide a host cell comprising the vector. means of solving the problem

[0017] According to one aspect of the present invention, the present invention provides a polynucleotide expressing RAD51 protein and Cas9 protein.

[0018] In the present invention, "RAD51 protein" is RAD51 It is a protein encoded by a gene that is known to play a role in assisting in the repair of DNA double strand damage.

[0019] In the present invention, "Cas9 protein" is a major protein component of the CRISPR / Cas9 system that forms a complex with crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA) to form an activated endonuclease or nickase.

[0020] Cas9 protein or gene information may be obtained from known databases such as, but is not limited to, GenBank of the NCBI (National Center for Biotechnology Information).

[0021] Additionally, the Cas9 protein may be linked to a protein transduction domain. The protein transduction domain may be a poly-arginine or HIV-derived TAT protein, but is not limited thereto. Furthermore, additional domains may be appropriately linked to the Cas9 protein by those skilled in the art, depending on the purpose.

[0022] Additionally, the Cas9 protein may include not only wild-type Cas9 but also variants of Cas9 such as inactivated Cas9 (dCas9) or Cas9 nickase. The inactivated Cas9 may be RFN (RNA-guided FokI Nuclease) with a FokI nuclease domain attached to dCas9, or dCas9 with a transcription activator or repressor domain attached, and the Cas9 nickase may be D10A Cas9 or H840A Cas9, but is not limited thereto.

[0023] The Cas9 protein of the present invention is not limited to its origin. For example, the Cas9 protein may be derived from Streptococcus pyogenes, Francisella novicida, Streptococcus thermophilus, Legionella pneumophila, Listeria innocua, or Streptococcus mutans.

[0024] In one embodiment of the present invention, the polynucleotide additionally comprises a guide RNA specific to a target sequence.

[0025] In the present invention, "guide RNA" may be composed of two RNAs, namely crRNA (CRISPR RNA) and tracrRNA (transactivating crRNA). Alternatively, it may be sgRNA (single guide RNA) prepared by the fusion of major portions of crRNA and tracrRNA. Additionally, the guide RNA may be a dual RNA comprising crRNA and tracrRNA.

[0026] RGEN, known as a third-generation gene scissors, may be composed of a Cas protein and dual RNA, or a Cas protein and sgRNA. The guide RNA may include one or more additional nucleotides at the 5' end of the crRNA of the sgRNA or dual RNA, and may be delivered into the cell in the form of RNA or DNA encoding said RNA.

[0027] In one embodiment of the present invention, the RAD51 protein is encoded by the nucleotide sequence of SEQ ID NO. 1.

[0028] In one embodiment of the present invention, the RAD51 protein comprises the amino acid sequence of SEQ ID NO. 2.

[0029] In one embodiment of the present invention, the polynucleotide is naked DNA or is contained in a gene carrier.

[0030] In one embodiment of the present invention, the gene carrier is a vector.

[0031] In one embodiment of the present invention, the vector is a plasmid vector, a cosmid vector, or a virus vector.

[0032] In the present invention, "vector" refers to an expression vector capable of expressing a target protein in a suitable host cell, comprising a gene construct that includes an essential regulatory element operably linked to enable the expression of a gene insert.

[0033] In the present invention, the term "operably linked" refers to a functional linkage between a nucleic acid expression regulatory sequence (e.g., a promoter, a signal sequence, or an array of transcription factor binding sites) and a nucleic acid sequence encoding a target protein to perform a general function. Accordingly, the regulatory sequence regulates the transcription and / or translation of the other nucleic acid sequence.

[0034] For example, by operably linking the sequences encoding the Cas9 and RAD51 proteins of the present invention to a promoter, the expression of said encoding sequences is placed under the influence or regulation of said promoter. Operable linkage with the recombinant vector can be prepared using gene recombination technology well known in the art, and site-specific DNA cleavage and linkage can be performed using enzymes, etc., generally known in the art.

[0035] The vector of the present invention may include signal sequences or leader sequences for membrane targeting or secretion in addition to expression regulatory elements such as promoters, operators, start codons, stop codons, polyadenylation signals, and enhancers, and may be manufactured in various ways depending on the purpose. The promoter of the vector may be constitutive or inducible. Additionally, the expression vector may include selectivity markers for selecting host cells containing the vector, and in the case of a replicable expression vector, may include a replication origin. The vector may self-replicate or be incorporated into host DNA.

[0036] The above vector may include a plasmid vector, a cosmid vector, or a virus vector, and specifically, it may be a virus vector.

[0038] i) Plasmid (vector)

[0039] A plasmid (vector) may be used as a carrier for carrying the polynucleotide of the present invention. It is preferable that the polynucleotide included in the vector be present in a suitable expression cassette. It is preferable that the polynucleotide in the expression cassette be operatively linked to a promoter.

[0040] In the present invention, the promoter coupled to the polynucleotide sequence is capable of operating in animal cells, preferably mammalian cells, more preferably human cells, to regulate the transcription of the nucleotide sequence, and includes a promoter derived from a mammalian virus and a promoter derived from the genome of a mammalian cell, such as, for example, a CMV (cytomegalovirus) promoter, an adenovirus late promoter, a vaccinia virus 7.5K promoter, an SV40 promoter, a tk promoter of HSV, an RSV promoter, an EF1 alpha promoter, a metallothionein promoter, a beta-actin promoter, a promoter of the human IL-2 gene, a promoter of the human IFN gene, a promoter of the human IL-4 gene, a promoter of the human lymphotoxin gene, and a promoter of the human GM-CSF gene, but is not limited thereto. More preferably, the promoter used in the present invention is a promoter derived from the IE (immediately early) gene of human CMV (hCMV) or an EF1 alpha promoter, and most preferably, it is a promoter / enhancer of the hCMV IE gene and a 5'-UTR (untranslated region) including the entire sequence of exon 1 up to just before the ATG start codon of exon 2.

[0041] The expression cassette used in the present invention may include a polyanenylation sequence, for example, a bovine growth hormone terminator (Gimmi, ER, et al., Nucleic Acids Res. 17:6983-6998 (1989)), an SV40-derived polyanenylation sequence (Schek, N, et al., Mol. Cell Biol. 12:5386-5393 (1992)), HIV-1 polyA (Klasens, BIF, et al., Nucleic Acids Res. 26:1870-1876 (1998)), beta-globin polyA (Gil, A., et al, Cell 49:399-406 (1987)), HSV TK polyA (Cole, CN and TP Stacy, Mol. Cell. Biol. 5:2104-2113 (1985)) or Polyomavirus polyA (Batt, D. B and GG Carmichael, Mol. Cell. Biol. 15:4783-4790 (1995)) is included, but not limited to.

[0043] ii) Retroviruses

[0044] Retroviruses are widely used as gene transfer vectors because they can insert their own genes into the host genome, carry large amounts of foreign genetic material, and have a broad spectrum of cells they can infect.

[0045] To construct a retroviral vector, the polynucleotide sequence of the present invention is inserted into the retroviral genome in place of the retroviral sequence to produce a non-replicating virus. To produce a virion, a packaging cell line is constructed containing the gag, pol, and env genes but lacking the LTR (long terminal repeat) and ψ sequences (Mann et al., Cell, 33:153-159 (1983)). When a recombinant plasmid containing the polynucleotide sequence, LTR, and ψ sequence of the present invention is introduced into said cell line, the ψ sequence enables the production of the RNA transcript of the recombinant plasmid, which is packaged into a virus, and the virus is released into the medium (Nicolas and Rubinstein "Retroviral vectors," In: Vectors: A survey of molecular cloning vectors and their uses, Rodriguez and Denhardt (eds.), Stoneham: Butterworth, 494-513 (1988)). A medium containing recombinant retroviruses is collected and concentrated to be used as a gene delivery system.

[0046] Gene delivery using second-generation retroviral vectors has been reported. Kasahara et al. (Science, 266:1373-1376 (1994)) produced a variant of the Molony-Murein leukemia virus and inserted an EPO (erythropoietin) sequence into the envelope region to produce a chimeric protein with novel binding characteristics. The polynucleotide sequence of the present invention can also be loaded into a retrovirus according to the same second-generation retroviral vector construction strategy.

[0048] iii) Adenovirus

[0049] Adenoviruses are widely used as gene delivery vectors due to their medium genome size, ease of manipulation, high titer, broad target cell range, and excellent infectivity. Both ends of the genome contain 100-200 bp inverted terminal repeats (ITRs), which are cis-elements essential for DNA replication and packaging. The E1 regions of the genome (E1A and E1B) encode proteins that regulate transcription and the transcription of host cell genes. The E2 regions (E2A and E2B) encode proteins involved in viral DNA replication.

[0050] Among the adenovirus vectors currently developed, non-replication adenoviruses lacking the E1 region are widely used. Meanwhile, the E3 region is removed in conventional adenovirus vectors to provide a site for the insertion of foreign genes (Thimmappaya, B. et al., Cell, 31:543-551 (1982); and Riordan, JR et al., Science, 245:1066-1073 (1989)). Therefore, it is preferable that the polynucleotide sequence of the present invention be inserted into the deleted E1 region (E1A region and / or E1B region) or the E3 region. Additionally, the nucleotide sequence may also be inserted into the deleted E4 region.

[0051] In this specification, the term "deletion" used in relation to viral genome sequences has the meaning of including not only the complete deletion of the sequence but also the partial deletion. Additionally, since adenoviruses can pack up to about 105% of the wild-type genome, they can additionally package about 2 kb (Ghosh-Choudhury et al., EMBOJ., 6:1733-1739 (1987)). Therefore, the aforementioned foreign sequence inserted into the adenovirus may additionally bind to the adenovirus genome.

[0052] Adenoviruses have 42 different serotypes and AF subgroups. Among these, adenovirus type 5, belonging to subgroup C, is the most preferred starting material for obtaining the adenovirus vector of the present invention. Biochemical and genetic information regarding adenovirus type 5 is well known. Foreign genes carried by adenoviruses are replicated in the same manner as episomes, and thus have very low genotoxicity to host cells. Therefore, gene therapy using an adenovirus gene delivery system is considered to be safe.

[0054] iv) AAV vector

[0055] Adeno-associated viruses (AAVs) are suitable as gene delivery systems of the present invention because they can infect non-dividing cells and have the ability to infect various types of cells. Detailed descriptions of the preparation and use of AAV vectors are disclosed in detail in U.S. Patents No. 5,139,941 and No. 4,797,368.

[0056] Studies on AAV as a gene delivery system are disclosed in LaFace et al, Viology, 162:483486 (1988), Zhou et al., Exp. Hematol. (NY), 21:928-933 (1993), Walsh et al, J. Clin. Invest., 94:1440-1448 (1994) and Flotte et al., Gene Therapy, 2:29-37 (1995).

[0057] Typically, an AAV virus is prepared by co-transforming a plasmid containing a target gene sequence with two adjacent AAV terminal repeats (McLaughlin et al., J. Virol., 62:1963-1973 (1988); and Samulski et al., J. Virol., 63:3822-3828 (1989)) and an expression plasmid containing a wild-type AAV coding sequence without terminal repeats (McCarty et al., J. Virol., 65:2936-2945 (1991)).

[0059] v) Other virus vectors

[0060] Other viral vectors can also be used to transport the polynucleotide sequence of the present invention into vivo. Puhlmann M. et al., Human Gene Therapy 10:649-657 (1999); Ridgeway, "Mammalian expression vectors," In: Vectors: A survey of molecular cloning vectors and their uses. Rodriguez and Denhardt, eds. Stoneham: Butterworth, 467-492 (1988); Baichwal and Sugden, "Vectors for gene transfer derived from animal DNA viruses: Transient and stable expression of transferred genes," In: Kucherlapati R, ed. Gene transfer. New York: Plenum Press, 117-148 (1986) and Coupar et al., Gene, 68:1-10 (1988)), lentivirus (Wang G. et al., J. Clin. Invest. 104(11):R55-62 (1999)) or Herpes simplex virus (Chamber R., et al., Vectors derived from Proc. Natl. Acad. Sci USA 92:1411-1415 (1995)) can also be used as a transport system capable of transporting the above polynucleotide into the cell.

[0062] vi) Liposomes

[0063] Liposomes are automatically formed by phospholipids dispersed in an aqueous phase. Examples of successfully transporting foreign DNA molecules into a cell via liposomes are disclosed in Nicolau and Sene, Biochim. Biophys. Acta, 721:185-190 (1982) and Nicolau et al., Methods Enzymol., 149:157-176 (1987). The liposomes containing the polynucleotide sequence of the present invention transport the polynucleotide sequence into the cell by interacting with the cell through mechanisms such as endocytosis, adsorption to the cell surface, or fusion with a plasma cell membrane.

[0064] In the present invention, when the polynucleotide sequence of the present invention is loaded onto a naked recombinant DNA molecule or a plasmid (vector), microinjection (Capecchi, MR, Cell, 22:479 (1980); and Harland and Weintraub, J. Cell Biol. 101:1094-1099 (1985)), calcium phosphate precipitation (Graham, FL et al., Virology, 52:456 (1973); and Chen and Okayama, Mol. Cell. Biol. 7:2745-2752 (1987)), electroporation (Neumann, E. et al., EMBO J., 1:841 (1982); and Tur-Kaspa et al., Mol. Cell Biol., 6:716-718 (1986)), liposome-mediated transfection (Wong, Polynucleotide sequences can be introduced into cells by TK et al., Gene, 10:87 (1980); Nicolau and Sene, Biochim. Biophys. Acta, 721:185-190 (1982); and Nicolau et al., Methods Enzymol., 149:157-176 (1987)), DEAE-dextran treatment method (Gopal, Mol. Cell Biol., 5:1188-1190 (1985)) and gene bombardment method (Yang et al., Proc. Natl. Acad. Sci., 87:9568-9572 (1990)).

[0065] When the polynucleotide sequence of the present invention is constructed based on a viral vector, the polynucleotide sequence can be delivered into a cell according to various viral infection methods known in the art. Infection of a host cell using a viral vector is described in the aforementioned cited literature.

[0067] vii) 2A peptide

[0068] 2A peptides are virus-derived oligopeptides with a structure consisting of approximately 18 to 22 amino acids that induce cleavage at the corresponding portion of the polypeptide formed during translation in eukaryotes. Consequently, two or more proteins can be produced from a single promoter. Depending on the type, examples include F2A (foot-and-mouth disease virus), E2A (equine rhinitis A virus), P2A (porcine teschovirus-1 2A), and T2A (thosea asigna virus 2A). When nucleotides corresponding to 2A on the genome are transcribed and translated to form a peptide, 2A, regardless of the specific type, generates a glycyl-prolyl peptide bond at the peptide's C-terminal, and ribosome skipping occurs in that region.

[0070] According to another aspect of the present invention, the present invention provides a genome editing composition comprising a polynucleotide expressing RAD51 protein and Cas9 protein.

[0071] In the present invention, the term "genome editing" refers to a technology capable of introducing targeted mutations into the genomic sequences of animal and plant cells, including human cells, by knocking out or knocking in specific genes, or by introducing mutations into non-coding DNA sequences that do not produce proteins. In a specific embodiment of the present invention, the genome editing refers to gene knock-out and may also be applied to knock-in.

[0072] In addition, DNA on the genome can be deleted, duplicated, inverted, replaced, or rearranged through genome editing.

[0073] In the present invention, "deletion" refers to a mutation that occurs due to the omission of a part of a chromosome or a part of a base on DNA.

[0074] In the present invention, "duplication" means that two or more of the same gene exist within the genome.

[0075] In the present invention, "inversion" refers to a portion of the genome being positioned upside down compared to the original genome.

[0076] In the present invention, "replacement" means that one nucleotide sequence is replaced with another (i.e., replaced with a sequence containing information), and does not necessarily mean that one polynucleotide is chemically or physically replaced with another polynucleotide.

[0077] In the present invention, "rearrangement" refers to a structural change that causes a change in the location and order of genes on a chromosome, and includes the insertion of transposable elements such as transposons. In addition, it may include the transformation of genetic information by base rearrangement within a DNA molecule.

[0078] In one embodiment of the present invention, the RAD51 protein and the Cas9 protein are encoded by separate nucleotide sequences.

[0079] In one embodiment of the present invention, the RAD51 protein and Cas9 protein are encoded by a single nucleotide sequence.

[0081] According to another aspect of the present invention, the present invention provides a host cell comprising a polynucleotide expressing RAD51 protein and Cas9 protein.

[0082] The term "host cell" refers to a eukaryotic or prokaryotic cell into which one or more DNA or vectors are "introduced into the cell," and should be understood to refer not only to a specific target cell but also to its offspring or potential offspring. Even if the offspring are not exactly identical to the parent cell due to mutation or environmental influence, they may still be included within the scope of the term as used herein. In the present invention, the host cell is one into which a polynucleotide (e.g., a viral vector) expressing the RAD51 protein and the Cas9 protein is introduced, from which a virus packaging a nucleotide encoding Cas9 can be obtained. Specifically, the virus can be obtained from a culture medium of the transformed cell or from a lysate of the cell.

[0083] The above cells may include prokaryotic cells such as E. coli, eukaryotic cells such as yeast, fungi, protozoa, higher plants, and insects, and mammalian cells such as CHO, HeLa, HEK293, and COS-1, but are not limited to the above examples.

[0084] In addition, it can be applied to all human cells, including somatic cells, germ cells, induced pluripotent stem cells, and adult stem cells.

[0085] The above somatic cells refer to all cells excluding germ cells obtainable from the bodies of embryos, children, and adults, and may include genetically modified cells derived from them. In addition, the above adult stem cells may include all adult stem cells obtainable from human embryos, newborns, and adults, as well as extraembryonic stem cells such as cord blood stem cells, placenta stem cells, Wharton's jelly stem cells, amniotic fluid stem cells, and amniotic epithelial cells, and genetically modified cells derived from them.

[0086] In addition, the above cells may be cultured cells (in vitro), grafts and primary cultures (in vitro and in vitro), and in vivo cells, and are not limited to cells commonly used in the industry.

[0087] However, the host cell of the present invention may be an isolated transformed cell excluding a human embryo.

[0088] In the present invention, "intracellular introduction" may utilize any method known in the art, and foreign DNA may be introduced into the cell by transfection or transduction. Transfection may be performed by various methods known in the art, such as calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroshock, microinjection, liposome fusion, lipofectamine, and protoplast fusion. Effects of the invention

[0090] The CRISPR Cas9 vector of the present invention RAD51When using the CRISPR-Cas9 vector of the present invention to express genes, the efficiency of gene editing increases by more than twofold, allowing for gene editing with higher efficiency and accuracy. As a result, by inactivating key genes associated with specific diseases with high efficiency, a model can be formed to study the disease, which can be helpful in disease research and the development of therapeutic agents. Brief explanation of the drawing

[0092] Figure 1 is a diagram showing the process of synthesizing the pLentiCRISPR v2-T2A-RAD51-sgGAPDH vector. Figure 2 illustrates the CRISPR-Cas9 vector introduction process to compare the efficiency of the CRISPR-Cas9 system according to the expression level of RAD51. Figure 2a. pLentiCRISPR v2-Cas9 and pLentiCRISPR v2-Cas9-T2A-Rad51; Figure 2b. pLentiCRISPR v2-Cas9 + pEF1α-RAD51; Figure 2c. pLentiCRISPR v2-Cas9 + pDonor and pLentiCRISPR v2-Cas9-T2A-Rad51 + pDonor, pLentiCRISPR v2-Cas9 + pEF1α-RAD51 + pDonor; Figure 2d. Schematic diagram of knock-out and knock-in genome editing. Figure 3 shows a comparison of the efficiency of a knock-out type GAPDH-targeting CRISPR-Cas9 system according to the expression level of RAD51 in HEK293T (human embryonic kidney 293t) cells. Figure 4 quantitatively shows the comparison of GADPH expression values ​​in a knock-out genome editing system according to the overexpression of RAD51 in HEK293T (human embryonic kidney 293t) cells. Significance probabilities were measured and analyzed using a paired, two-tailed Student's t-test (*p < 0.05, **p < 0.01, ***p < 0.001). Figure 5 shows a comparison of the efficiency of a knock-in GAPDH-targeting CRISPR-Cas9 system according to the expression level of RAD51 in HEK293T (human embryonic kidney 293t) cells. Figure 6 quantitatively shows the comparison of GADPH expression values ​​in a knock-in genome editing system according to the overexpression of RAD51 in HEK293T (human embryonic kidney 293t) cells. Significance probabilities were measured and analyzed using a paired, two-tailed Student's t-test (*p < 0.05, **p < 0.01, ***p < 0.001). Figure 7 is a figure showing a pLentiCRISPR v2 vector map that can insert an sgRNA sequence complementary to a specific sequence of a target gene. Figure 8 shows the pLentiCRISPR v2-sgGAPDH-T2A-RAD51 vector map with Rad51 CDS inserted. Specific details for implementing the invention

[0093] The present invention will be described in more detail below through examples. These examples are intended solely to explain the invention more specifically, and it will be obvious to those skilled in the art that the scope of the invention is not limited by these examples according to the gist of the invention.

[0095] Examples

[0097] Throughout this specification, "%" used to indicate the concentration of a particular substance is (weight / weight) % for solid / solid, (weight / volume) % for solid / liquid, and (volume / volume) % for liquid / liquid, unless otherwise noted.

[0099] Example 1. RAD51 Production of a CRISPR-Cas9 plasmid vector with inserted genes

[0100] The inventors of the present invention RAD51 A CRISPR-Cas9 plasmid vector with the inserted gene was constructed as follows. As shown in Figure 1, first, RAD51 To express the gene, the T2A peptide sequence and the Rad51 CDS sequence were obtained. The Rad51 CDS sequence is shown in SEQ ID NO. 1, and the T2A peptide sequence is shown in SEQ ID NO. 3. As the CRISPR-Cas9 plasmid, the pLentiCRISPR v2 plasmid (Addgene, USA) containing the Cas9 gene was used. The vector map of the pLentiCRISPR v2 plasmid is shown in Fig. 5. After cloning the T2A peptide and Rad51 CDS sequences following the Cas9 gene sequence present in the plasmid, an sgRNA sequence for the target gene was synthesized, and an sgRNA sequence complementary to a specific sequence of the target gene was inserted into the U6 promoter using the BsmB I enzyme site. In the embodiment of the present invention, the sgRNA used a specific sequence targeting the GAPDH gene (SEQ ID NO. 5).

[0102] Example 2. RAD51 Verification of the efficiency of gene-inserted CRISPR-Cas9 plasmid vector

[0103] The inventors of the present invention RAD51In order to measure the change in efficiency of the CRISPR-Cas9 system due to gene insertion, the [prepared in Example 1] RAD51 A CRISPR-Cas9 plasmid with the inserted gene (designated as pLentiCRISPR v2-RAD51), and RAD51 The editing efficiency in knock-out and knock-in genome editing was confirmed by introducing and expressing a CRISPR-Cas9 plasmid (designated as pLentiCRISPR v2) without gene insertion into human cell lines, respectively.

[0105] Example 2.1. Knock-out method dielectric correction efficiency

[0106] Introduction of CRISPR-Cas9 vectors for the knockout method

[0107] The process for observing the correction efficiency in knock-out genome editing is as follows.

[0108] First, human cell line HEK293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C and 5% CO2. A mixture containing 3 µg of each of the pLentiCRISPR v2-RAD51 and pLentiCRISPR v2 plasmids and 20 µg of the cationic polymer PEI (polyethyleneimine) was added to the Dulbecco's modified Eagle's medium (DMEM) in which the cell lines were cultured to induce transformation. Subsequently, each cell line was cultured at 37°C and 5% CO2 for 24 hours, and only cells containing pLentiCRISPR v2-RAD51 and pLentiCRISPR v2 plasmids were harvested after treatment with 1 μg / ml of antibiotic (puromycin) (Fig. 2a). In addition, the individual vector containing RAD51 (pEF1α-RAD51) was re-transformed into cells containing pLentiCRISPR v2 plasmid (plasmid 1 μg, PEI 10 μg) and harvested after 24 hours, thereby allowing for a comparison of gene editing efficiency according to the expression mode of RAD51 (Fig. 2b).

[0110] Confirmation of knockout-style genome editing efficiency based on GAPDH expression levels

[0111] The efficiency of the knock-out genome editing method was confirmed by comparing the expression levels of GAPDH using a western blot.

[0112] As a result, as shown in Fig. 3a, the expression level of GAPDH decreased due to the influence of the CRIPSR-Cas9 system in cells inserted with the pLentiCRISPR v2-sgGAPDH vector (pLentiCRISPR v2) compared to the control group (HEK293T normal). At this time, compared to the control group (HEK293T normal) and cells introduced only with the pLentiCRISPR v2-sgGAPDH vector (pLentiCRISPR v2), the expression level of GAPDH decreased even further in cells with the introduction of the CRISPR v2-T2A-RAD51 vector, which overexpressed RAD51 (pLentiCRISPR v2-RAD51). This shows the same trend as the decrease in GAPDH expression observed in cells in which the pEF1α-RAD51 plasmid was introduced individually along with the pLentiCRISPR v2-sgGAPDH vector in Fig. 3b.

[0113] In addition, GADPH expression values ​​were compared and quantitatively expressed in Figures 4a and 4b. When comparing the expression levels of GAPDH under each condition, cells inserted with the pLentiCRISPR v2-sgGAPDH vector (pLentiCRISPR v2) showed an expression level of approximately 47% when the GAPDH expression level of the control group was set to 100%. However, cells introduced with the pLentiCRISPR v2-T2A-RAD51 vector of the present invention (pLentiCRISPR v2-RAD51) showed an expression level of approximately 25% of GAPDH. That is, in cells inserted with the pLentiCRISPR v2-sgGAPDH vector (pLentiCRISPR v2), the expression level of GAPDH was reduced by about 53% compared to the control group, and in cells introduced with the pLentiCRISPR v2-T2A-RAD51 vector (pLentiCRISPR v2-RAD51), the expression level of GAPDH was reduced by about 75% compared to the control group and by about 22% compared to cells introduced with the pLentiCRISPR v2-sgGAPDH vector (pLentiCRISPR v2) (Fig. 4a).

[0114] The above results were similarly observed in cells expressing RAD51 using the individual vectors of Fig. 4b. Cells inserted only with the pLentiCRISPR v2-sgGAPDH plasmid (pLentiCRISPR v2) showed an expression level of approximately 56% of GAPDH. However, cells introduced with both the pLentiCRISPR v2-sgGAPDH and RAD51 vectors showed an expression level of approximately 26% of GAPDH. That is, when the GAPDH expression level of the control group was set to 100%, the expression level of GAPDH in cells with the pLentiCRISPR v2-sgGAPDH vector inserted (pLentiCRISPR v2) was reduced by about 44% compared to the control group, and in cells with the RAD51 vector separately introduced (pLentiCRISPR v2 + pEF1α-RAD51), the expression level of GAPDH was reduced by about 74% compared to the control group, and by about 30% compared to cells with the pLentiCRISPR v2-sgGAPDH vector introduced (pLentiCRISPR v2).

[0115] Consequently, this means that the efficiency of the knock-out gene editing technology of the CRISPR-Cas9 system can be increased using only the CRISPR v2-T2A-RAD51 single vector, without the need to insert the RAD51 plasmid into cells individually.

[0117] Example 2.2. Knock-in method dielectric correction efficiency

[0118] Introduction of CRISPR-Cas9 vectors for the knock-in method

[0119] To compare the editing efficiency in the knock-in method of genome editing, the same procedure as the knock-out method of Example 2.1 was performed. In addition to the above method, 2 μg of a donor plasmid having a homology arm for the GAPDH gene was transformed into cell lines containing pLentiCRISPR v2-RAD51, pLentiCRISPR v2, and pLentiCRISPR v2 + pEF1α-RAD51, respectively, using 15 μg of PEI. These were harvested 24 hours after transformation (Fig. 2c).

[0121] Confirmation of knock-in genome editing efficiency based on GAPDH expression levels

[0122] The efficiency of genome editing was confirmed by comparing the expression levels of GAPDH using Western blot for knock-in genome editing.

[0123] As can be seen in Fig. 5a, the results showed that the efficiency of the knock-in gene editing technique using the CRISPR v2-T2A-RAD51 vector also increased, similar to the efficiency of the knock-out gene editing technique described above. This also shows the same trend as in the cells in which the pEF1α-RAD51 plasmid was individually introduced along with the pLentiCRISPR v2-sgGAPDH vector in Fig. 5b.

[0124] The quantitative results for this are shown in Figures 6a and 6b. As a result, cells inserted with the pLentiCRISPR v2-sgGAPDH vector (pLentiCRISPR v2) showed an expression level of approximately 40% of GAPDH. However, cells introduced with the pLentiCRISPR v2-T2A-RAD51 vector of the present invention (pLentiCRISPR v2-RAD51) showed an expression level of approximately 24% of GAPDH. That is, in cells inserted with the pLentiCRISPR v2-sgGAPDH vector (pLentiCRISPR v2), the expression level of GAPDH was reduced by about 60% compared to the control group (100%), and in cells introduced with the pLentiCRISPR v2-T2A-RAD51 vector (pLentiCRISPR v2-RAD51), the expression level of GAPDH was reduced by about 76% compared to the control group and by about 16% compared to cells introduced with the pLentiCRISPR v2-sgGAPDH vector (pLentiCRISPR v2) (Fig. 6a).

[0125] The same results were observed in cells expressing RAD51 using individual vectors in Fig. 6b. Cells inserted only with the pLentiCRISPR v2-sgGAPDH plasmid (pLentiCRISPR v2) showed an expression level of approximately 58% of GAPDH. However, cells introduced with both the pLentiCRISPR v2-sgGAPDH and RAD51 vectors showed an expression level of approximately 36% of GAPDH. That is, when the GAPDH expression level of the control group was set to 100%, the expression level of GAPDH in cells with the pLentiCRISPR v2-sgGAPDH vector inserted (pLentiCRISPR v2) was reduced by about 42% compared to the control group, and in cells with the RAD51 vector separately introduced (pLentiCRISPR v2 + pEF1α-RAD51), the expression level of GAPDH was reduced by about 64% compared to the control group, and by about 22% compared to cells with the pLentiCRISPR v2-sgGAPDH vector introduced (pLentiCRISPR v2).

[0126] This once again demonstrated that the efficiency of knock-in gene editing technology using the CRISPR v2-T2A-RAD51 vector is similar to that of knock-out gene editing technology.

[0127] Therefore, from the results of the knock-out and knock-in genome editing methods described above, it was found that inducing the overexpression of the RAD51 protein within a single plasmid vector increased the efficiency of the gene editing technology by approximately twofold compared to when editing was applied to a target gene using the conventional CRISPR-Cas9 system.

Claims

Claim 1 delete Claim 2 delete Claim 3 delete Claim 4 delete Claim 5 delete Claim 6 delete Claim 7 A genome editing composition comprising a polynucleotide expressing a RAD51 protein and a Cas9 protein, wherein the RAD51 protein is encoded by the nucleotide sequence of SEQ ID NO. 1; the RAD51 protein comprises the amino acid sequence of SEQ ID NO. 2; and the RAD51 protein and the Cas9 protein are encoded by a single nucleotide sequence, wherein the gene editing is a gene knockout. Claim 8 A genome editing composition according to claim 7, wherein the polynucleotide is naked DNA or is contained in a gene carrier. Claim 9 In claim 8, the gene carrier is a vector, a composition for genome editing. Claim 10 delete Claim 11 delete

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