N-glycosylated mutant rice, method for producing the same, and method for producing protein-producing rice using the same

Abstract

The present invention relates to N-glycosylation mutant rice (Oryza sativa), a method for producing the same, and a method for producing rice for protein production using the same. Mutant cell lines for a total of eight genes involved in the plant-specific N-glycosylation process have been obtained, and the possibility of producing medical proteins from these has been confirmed, so they can be effectively used for protein production.

Description

Technical Field

[0001] The present invention was carried out under the support of the Rural Development Administration with the project unique number 1395062825 and the detailed project number PJ013659012020. The research management specialized agency for the said project is the Rural Development Administration, the name of the research project is "Next Generation Bio Green 21 Project", the name of the research topic is "Development of rice cell lines for producing human-like glycoproteins and production of medical proteins", the competent agency is the Industry-Academia Cooperation Group of Sogang University, and the research period is from March 1, 2018 to December 31, 2020.

[0002] This patent application claims priority to Korean Patent Application No. 10-2020-0090189, which was filed with the Korean Intellectual Property Office on July 21, 2020, and the disclosure of the said patent application is incorporated herein by reference.

[0003] The present invention relates to an N-glycosylation mutant rice (Oryza sativa), a method for producing the same, and a method for producing rice for protein production using the same. More specifically, the present invention relates to a technique for establishing a rice cell line capable of producing medical proteins by correcting a total of eight genes involved in the plant-specific N-glycosylation process to remove plant-specific glycosylation.

Background Art

[0004] Post-translational modification of proteins in all eukaryotic organisms including plants is an essential process for the final activity of proteins. In particular, the N-glycosylation process is a modification process for membrane proteins and secreted proteins, and is a process of attaching a special sugar chain to an asparagine residue in the motif of asparagine (ASN)-X (amino acid excluding proline)-serine or threonine (Ser / Thr) in the protein sequence. Such a sugar chain polymerization process has an important influence on the activity, structure, action position, substrate recognition, and stability of glycoproteins.

[0005] The N-glycosylation process of proteins, which begins in the endoplasmic reticulum, occurs commonly in all eukaryotes studied, with the attachment of oligomannosidic N-glycan forms where mannose is exposed. These glycans are then converted into more complex forms in the Golgi apparatus, with various sugars such as fucose, xylose, and galactose being attached during this process. This type of process is called complex N-glycosylation.

[0006] This complex N-glycosylation process differs slightly from organism to organism. In particular, plants differ from animals in that they specifically attach α1,3 fucose and β1,2 xylose, and then, in the final conversion process that occurs in the trans-Golgi network, α1,4 fucose and β1,3 galactose are attached.

[0007] Receptors that sense environmental stimuli and stress are expected to exist mostly in the form of glycoproteins. Therefore, research on glycosylation is needed to understand the mechanism of action of such proteins, and in turn, is expected to help in understanding the mechanism of action of plants in response to stress.

[0008] To date, research on the function of N-glycosylation genes in plants has mainly been conducted in Arabidopsis thaliana. Mutations of genes involved in N-glycosylation in the endoplasmic reticulum have been observed to cause deformation of the cell wall structure and inhibition of root growth, as well as increased sensitivity to stress.

[0009] Mutants to the gene involved in the combined N-glycosylation between Arabidopsis thaliana and tobacco did not exhibit any significant phenotypic changes, and their survival was not greatly impaired.

[0010] In contrast, mutations in complex N-glycosylation genes in rice, a monocotyledonous model plant, exhibited diverse phenotypes at the developmental stage, particularly showing increased sensitivity to salt stress treatment.

[0011] These results suggest that while the structure of complex N-linked glycans is not essential for life processes or stress responses in Arabidopsis thaliana and tobacco, in rice (Oryza sativa), the complex glycan structure can perform important, yet unknown, functions, such as being more sensitive to ER stress. However, to date, functional studies of these genes and their stress-related studies in rice remain in their early stages. Furthermore, previous studies have shown that mutations in N-glycosylation-related genes in rice have a fatal impact on life processes, making functional studies of these genes very difficult.

[0012] While the N-glycosylation process of proteins is known to have a significant impact on protein function and stability, research on N-glycosylation in plants is currently insufficient. It is expected that most receptors that sense environmental stimuli and stress, as well as proteins involved in cell wall construction, exist in the form of glycoproteins in plants. Therefore, glycosylation-related research is needed to understand the mechanisms of action of such proteins, and is also expected to help understand the mechanisms by which plants respond to stress.

[0013] The market for biopharmaceuticals is rapidly expanding globally, and in South Korea, it is gaining attention as one of the key businesses of the next generation. While biopharmaceuticals have traditionally been produced using animal cells, or bacteria and yeast, these methods suffer from various chronic problems, including high manufacturing costs. Therefore, attempts are being made to compensate for these shortcomings by producing medical proteins using plant systems.

[0014] However, N-glycosylation in mammals, including humans, manifests differently than in plants. Therefore, the development of human-type N-glycosylated plant materials that overcome these problems is an extremely urgent matter. [Overview of the project] [Problems that the invention aims to solve]

[0015] Therefore, in order to investigate the function of plant-specific N-glycosylation processes in rice (Oryza sativa) on environmental stress, the inventors obtained mutant cell lines for a total of eight genes involved in such processes and confirmed that these can be utilized as plant systems for the production of target proteins.

[0016] Therefore, the object of the present invention is to provide a vector for correcting N-glycosylation genes in rice, which contains a guide RNA consisting of one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 1 to 16.

[0017] Another object of the present invention is to provide N-glycosylated mutant rice transformed with a vector containing a guide RNA consisting of one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 1 to 16.

[0018] Another object of the present invention is to provide a method for producing N-glycosylated mutant rice, which includes a gene correction step of transforming rice with a rice N-glycosylation gene correction vector containing a guide RNA consisting of one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 1 to 16.

[0019] Another object of the present invention is to provide a trastuzumab production composition comprising a trastuzumab light chain synthesis gene consisting of the nucleotide sequence of SEQ ID NO: 44 and a trastuzumab heavy chain synthesis gene consisting of the nucleotide sequence of SEQ ID NO: 45, which are codon-optimized to express trastuzumab (TMab) in rice.

[0020] Another object of the present invention is to provide a trastuzumab expression vector comprising a trastuzumab light chain synthesis gene consisting of the nucleotide sequence of SEQ ID NO: 44 and a trastuzumab heavy chain synthesis gene consisting of the nucleotide sequence of SEQ ID NO: 45, which are codon-optimized to express trastuzumab in rice.

[0021] A further object of the present invention is a vector for correcting the N-glycosylation gene of rice, comprising a guide RNA consisting of one or more base sequences selected from the group consisting of SEQ ID NOs: 1 to 16; and A vector for expressing trastuzumab, comprising a trastuzumab light chain synthetic gene consisting of the base sequence of SEQ ID NO: 44 and a trastuzumab heavy chain synthetic gene consisting of the base sequence of SEQ ID NO: 45, which are codon-optimized so that trastuzumab is expressed in rice To provide rice for producing trastuzumab transformed with the above.

[0022] Another further object of the present invention is to provide a method for producing rice for protein production, comprising the following steps.

[0023] A gene correction step of transforming rice with a vector for correcting the N-glycosylation gene of rice, comprising a guide RNA consisting of one or more base sequences selected from the group consisting of SEQ ID NOs: 1 to 16; and A gene introduction step of transforming rice with a vector containing a gene encoding a target protein.

[0024] Another further object of the present invention relates to the use of plant-specific N-glycosylation mutant rice for protein production.

Means for Solving the Problems

[0025] The present invention relates to N-glycosylation (N-glycosylation) mutant rice (Oryza sativa), a method for producing the same, and a method for producing rice for protein production using the same. The N-glycosylation mutant rice according to the present invention can be utilized as a plant system for producing a target protein.

[0026] The present inventor tried to help the understanding of N-glycosylation in plants by using mutant cell lines of genes involved in plant-specific glycosylation processes to study the effects of the defects in the functions of such genes on life phenomena at the level of plant cells.

[0027] Therefore, in order to investigate the function of the plant-specific N-glycosylation process in rice on environmental stress, a total of eight genes involved in such a process were mutated using CRISPR-Cas9 technology. As a result, functional mutant cell lines of eight genes (β1,2-XylT, α1,3-FucT, β1,3-GalT, α1,4-FucT, HEXO1, HEXO2, HEXO3, and HEXO4) and mutant cell lines of four genes (α1,3-FucT, β1,2-XylT, β1,3-GalT, and α1, and 4-FucT) were respectively obtained, and it was confirmed that these could be utilized as a plant system for the production of target proteins.

[0028] Using such a cell line system in which plant-specific glycosylation is removed, it is expected that the stress mechanism at the cell level can be more effectively carried out, and a protein production system can be constructed and used as a platform for the production of useful proteins such as protein pharmaceuticals.

[0029] Hereinafter, the present invention will be described in more detail.

[0030] One aspect of the present invention is a guide RNA consisting of one or more base sequences selected from the group consisting of SEQ ID NOs: 1 to 16.

[0031] In the present invention, the guide RNA can target one or more genes selected from the group consisting of β1,2-XylT, α1,3-FucT, β1,3-GalT, α1,4-FucT, HEXO1, HEXO2, HEXO3, and HEXO4.

[0032] The above genes correspond to the genes involved in the plant-specific glycosylation process.

[0033] In the present invention, the guide RNA may be a polycistronic guide RNA designed to express two or more base sequences selected from the group consisting of SEQ ID NOs: 1 to 16.

[0034] In this specification, the term "polycistronic" refers to a state in which a single transcription unit of mRNA contains multiple amino acid sequences (cistrons) defined by translation initiation and termination signals. In contrast, when only one cistron is present, it is called a monocistron.

[0035] In one embodiment of the present invention, a polycistronic guide RNA was synthesized and used, designed to express eight nucleotide sequences selected from the group consisting of SEQ ID NOs: 1 to 16. The eight nucleotide sequences may be selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, and 15, or from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, and 8, but are not limited to these.

[0036] Another aspect of the present invention is a vector for correcting the N-glycosylation gene in rice, comprising a guide RNA consisting of one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 1 to 16.

[0037] The term "vector" refers to a means of expressing a target gene in a host cell. It means a DNA fragment(s) or nucleic acid molecule that is transmitted into a cell, and can be replicated and reproduced independently of the host cell.

[0038] An "expression vector" refers to a recombinant DNA molecule containing the target coding sequence and the appropriate nucleic acid sequences essential for expressing the coding sequence operably linked in a particular host organism. An expression vector may contain one or more origins of replication, promoters, selection markers, enhancers, termination signals, and polyadenylation sequences available in eukaryotic cells. An expression vector may generally be derived from plasmid or viral DNA, or may contain elements of both. Thus, an expression vector refers to recombinant DNA or RNA constructs, such as plasmids, phages, recombinant viruses, or other vectors that, upon introduction into a suitable host cell, induce the expression of cloned DNA. Suitable expression vectors are well known to those skilled in the art and include those that are replicable in eukaryotic and / or prokaryotic cells, those that remain as episomes, and those that are integrated into the host cell genome.

[0039] Known vectors include pROKII, pBI76, pET21, pSK(+), pLSAGPT, pUC, and pGEM. However, these recombinant vectors are preferably recombinant plant expression vectors.

[0040] A desirable example of a plant expression vector is a Ti-plasmid vector, which, when present in a suitable host such as Agrobacterium tumefaciens, can transfer a portion of itself, the so-called T-region, to plant cells. Other types of Ti-plasmid vectors (see EP0116718B1) are currently used to transfer hybrid DNA sequences to plant cells or to protoplasms that can produce new plants by appropriately inserting the hybrid DNA into the plant genome. A particularly desirable form of Ti-plasmid vector is the so-called binary vector, as disclosed in EP0120516B1 and U.S. Patent No. 4,940,838.

[0041] Other suitable vectors that can be used to introduce the DNA according to the present invention into a plant host can be selected from viral vectors such as double-stranded plant viruses (e.g., CaMV) and single-stranded viruses, geminiviruses, etc., such as incomplete plant viral vectors. The use of such vectors may be particularly advantageous when the plant host is suitable and difficult to transform. The expression vector preferably includes one or more selective markers. The markers are typically nucleic acid sequences with properties that can be selected by chemical means, and include all genes that can distinguish transformed cells from non-transformed cells. Examples include, but are not limited to, herbicide resistance genes such as glyphosate or phosphinotricin, and antibiotic resistance genes such as kanamycin, G418, bleomycin, hygromycin, and chloramphenicol.

[0042] In one embodiment of the present invention, the promoter of the plant expression vector may be, but is not limited to, CaMV35S, actin, ubiquitin, pEMU, MAS, or histone promoters. The term "promoter" refers to the upstream region of DNA from a structural gene and to the DNA molecule to which RNA polymerase binds for transcription initiation. A "plant promoter" is a promoter that can initiate transcription in plant cells. A "constitutive promoter" is a promoter that is active under most environmental conditions and developmental stages or cell differentiation. Constitutive promoters may be desirable in the present invention because the selection of transformants can be carried out by various combinations at various stages. Therefore, constitutive promoters do not limit selectivity. Furthermore, the vector can use conventional terminators, including, but not limited to, nopaline synthase (NOS), rice α-amylase RAmy1A terminator, phaseoline terminator, and the octopine gene terminator of Agrobacterium tumefaciens. Regarding the necessity of terminators, it is generally known that such regions increase the reliability and efficiency of transcription in plant cells. Therefore, the use of terminators can be very desirable in the context of this invention.

[0043] The recombinant vector can be constructed using prokaryotic or eukaryotic cells as a host. For example, if the vector used is an expression vector and prokaryotic cells are used as the host, a potent promoter (e.g., pL) that can promote transcription can be used. λThe vector typically contains promoters (such as CMV promoters, trp promoters, lac promoters, tac promoters, and T7 promoters), ribosome binding sites for initiating sequencing, and transcription / sequencing termination sequences. When eukaryotic cells are used as the host, the origins of replication that act in eukaryotic cells and contained within the vector include, but are not limited to, f1, SV40, pMB1, adeno, AAV, and BBV origins. In addition, the transcription termination sequence usually contains a polyadenylated sequence.

[0044] Plant transformation refers to any method of transferring DNA to a plant. Such transformation methods do not necessarily require a regeneration and / or a culture period. Plant species transformation is now common to plant species that include both dicotyledonous and monocotyledonous plants. In principle, any transformation method can be used to introduce the hybrid DNA according to the present invention into suitable progenitor cells. The methods include the calcium / polyethylene glycol method for protoplasm (Krens, FA et al., 1982, Nature 296, 72-74; Negrutiu I. et al., 1987, Plant Mol. Biol. 8, 363-373), electroporation of protoplasm (Shillito RD et al., 1985 Bio / Technol. 3, 1099-1102), microinjection into plant material (Crossway A. et al., 1986, Mol. Gen. Genet. 202, 179-185), and particle impaction of various plant materials (DNA or RNA coated) (Klein TM et al., 1987, Nature 327, 70) Agrobacterium tumefaciens-mediated gene transfer by plant infiltration or transformation of mature pollen or microspores can be appropriately selected from (incomplete) viral infection (EP0301316), etc. The preferred method according to the present invention includes Agrobacterium-mediated DNA transfer.

[0045] The "plant cells" used in plant transformation may be cultured cells, cultured organisms, cultured bodies, or whole plants, preferably in the form of cultured cells, cultured organisms, or cultured bodies, and more preferably in the form of cultured cells.

[0046] "Plants" include differentiated or differentiated plant structures, such as, but not limited to, roots, stems, leaves, pollen, seeds, tumor structures, and various cellular forms used for culture, namely single cells, protoplasts, buds, and callus structures. Plant structures may be in planta, in organ culture, in structure culture, or in cell culture.

[0047] The method for selecting the transformed host cells can be easily carried out by a method widely known in the art, utilizing the phenotype expressed by the selective label. For example, if the selective label is a specific antibiotic resistance gene, the transformants can be easily selected by culturing them in a medium containing the antibiotic.

[0048] In the present invention, the guide RNA can target one or more genes selected from the group consisting of β1,2-XylT, α1,3-FucT, β1,3-GalT, α1,4-FucT, HEXO1, HEXO2, HEXO3, and HEXO4.

[0049] In the present invention, the guide RNA can be a polycistronic guide RNA designed to express two or more base sequences selected from the group consisting of SEQ ID NOs: 1 to 16.

[0050] Another aspect of the present invention is an N-glycosylated mutant rice transformed with a vector containing a guide RNA consisting of one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 1 to 16.

[0051] In the present invention, the guide RNA may target one or more genes selected from the group consisting of β1,2-XylT, α1,3-FucT, β1,3-GalT, α1,4-FucT, HEXO1, HEXO2, HEXO3, and HEXO4.

[0052] In the present invention, the guide RNA may be a polycistronic guide RNA designed to express two or more base sequences selected from the group consisting of SEQ ID NOs: 1 to 16.

[0053] In the present invention, rice may be selected from the group consisting of cells, callus, seeds, and adult plants, and may, for example, be a cellular plant, but is not limited thereto.

[0054] Another aspect of the present invention is a method for producing N-glycosylated mutant rice, comprising a gene correction step of transforming rice with a rice N-glycosylation gene correction vector containing a guide RNA consisting of one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 1 to 16.

[0055] In the present invention, the guide RNA can target one or more genes selected from the group consisting of β1,2-XylT, α1,3-FucT, β1,3-GalT, α1,4-FucT, HEXO1, HEXO2, HEXO3, and HEXO4.

[0056] In the present invention, the guide RNA may be a polycistronic guide RNA designed to express two or more base sequences selected from the group consisting of SEQ ID NOs: 1 to 16.

[0057] In the present invention, rice may be selected from the group consisting of cells, callus, seeds, and adult plants, and may, for example, be cells, but is not limited thereto.

[0058] Another aspect of the present invention is a trastuzumab production composition comprising a trastuzumab light chain synthesis gene consisting of the nucleotide sequence of SEQ ID NO: 44 and a trastuzumab heavy chain synthesis gene consisting of the nucleotide sequence of SEQ ID NO: 45, which have been codon-optimized to express trastuzumab (TMab) in rice.

[0059] In this specification, the term "codon optimization" refers to the optimization of codon usage for each species in order to optimize protein synthesis. Since the optimal codons differ depending on the species, codon optimization is also necessary when you want to efficiently express a desired protein in another species. For example, when expressing a human protein in Intestinal Bacteria, changing the codons that encode the amino acids to those mainly used by Intestinal Bacteria can make the protein more efficiently expressed.

[0060] Another aspect of the present invention is a trastuzumab expression vector comprising a trastuzumab light chain synthesis gene consisting of the nucleotide sequence of SEQ ID NO: 44 and a trastuzumab heavy chain synthesis gene consisting of the nucleotide sequence of SEQ ID NO: 45, which are codon-optimized to express trastuzumab in rice.

[0061] Another aspect of the present invention is a vector for correcting N-glycosylation genes in rice, comprising a guide RNA consisting of one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 1 to 16; and A trastuzumab expression vector containing a trastuzumab light chain synthesis gene consisting of the nucleotide sequence of SEQ ID NO: 44 and a trastuzumab heavy chain synthesis gene consisting of the nucleotide sequence of SEQ ID NO: 45, which have been codon-optimized to express trastuzumab in rice. This is rice transformed for trastuzumab production.

[0062] In the present invention, the guide RNA can target one or more genes selected from the group consisting of β1,2-XylT, α1,3-FucT, β1,3-GalT, α1,4-FucT, HEXO1, HEXO2, HEXO3, and HEXO4.

[0063] In the present invention, the guide RNA may be a polycistronic guide RNA designed to express two or more base sequences selected from the group consisting of SEQ ID NOs: 1 to 16.

[0064] In the present invention, rice may be selected from the group consisting of cells, callus, seeds, and adult plants, and may, for example, be cells, but is not limited thereto.

[0065] Another aspect of the present invention is to provide a method for producing rice for protein production, comprising the following steps:

[0066] A gene correction step in which rice is transformed with a rice N-glycosylation gene correction vector containing a guide RNA consisting of one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 1 to 16; and This is the gene transfer stage in which rice plants are transformed with a vector containing the gene coding for the target protein.

[0067] In this invention, the target protein may be trastuzumab.

[0068] In the present invention, the guide RNA can target one or more genes selected from the group consisting of β1,2-XylT, α1,3-FucT, β1,3-GalT, α1,4-FucT, HEXO1, HEXO2, HEXO3, and HEXO4.

[0069] In the present invention, the guide RNA can be a polycistronic guide RNA designed to express two or more base sequences selected from the group consisting of SEQ ID NOs: 1 to 16.

[0070] In the present invention, rice may be selected from the group consisting of cells, callus, seeds, and adult plants, and may, for example, be cells, but is not limited thereto. [Effects of the Invention]

[0071] This invention relates to N-glycosylation mutant rice (Oryza sativa), a method for producing the same, and a method for producing protein-producing rice using the same. Mutant cell lines have been obtained for a total of eight genes involved in the plant-specific N-glycosylation process, and the possibility of producing medical proteins from these cells has been confirmed, making it possible to effectively utilize them for protein production. [Brief explanation of the drawing]

[0072] [Figure 1] Figure 1 is a photograph of the results of an in vitro cleavage experiment to confirm the activity levels of sgRNA (singleguideRNA) and Cas9. [Figure 2a] Figure 2a is a schematic diagram of the pSK437 vector, which is a CRISPR-Cas9 vector. [Figure 2b] Figure 2b is a schematic diagram of the pSK438 vector, which is a CRISPR-Cas9 vector. [Figure 3] Figure 3 shows photographs of single cells (left) or cell populations derived from single cells (right) obtained from suspension culture of the #1-12-20-11 callus line. [Figure 4] Figure 4 shows photographs of the immunoblot results for α1,3-fucose and β of the acquired single cell line. [Figure 5] Figure 5 shows a photograph of the results of confirming the expression of trastuzumab (TMab) in a single cell line with a plant-specific glycosylation gene mutation. [Modes for carrying out the invention]

[0073] The present invention relates to N-glycosylated mutant rice (Oryza sativa) transformed with a vector containing a guide RNA consisting of one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 1 to 16.

[0074] Next, the present invention will be described in more detail by the following examples. However, these examples are merely illustrative of the present invention, and the scope of the present invention is not limited by these examples.

[0075] Throughout this specification, the percentages used to indicate the concentration of a particular substance refer to (weight / weight)%, (weight / volume)%, and (volume / volume)%, respectively, for solid / solid, unless otherwise specified.

[0076] Example 1: Design of target sequence and construction of CRISPR-Cas9 vector To create mutant rice (Oryza sativa) cell lines for eight genes (β1,2-XylT, α1,3-FucT, β1,3-GalT, α1,4-FucT, HEXO1, HEXO2, HEXO3, HEXO4) and four genes (β1,2-XylT, α1,3-FucT, β1,3-GalT, α1,4-FucT) involved in plant-specific N-glycosylation and N-linked glycosylation processes, we used a CRISPR-PgRNA design tool to select two optimal target sequences per gene at the exon region of each gene, which were then used to construct CRISPR-Cas9 vectors.

[0077] We confirmed whether sgRNA (single guide RNA)-Cas9 complexes, selected by PCR amplification of target sites of eight genes, could actually cleave DNA through in vitro cleavage experiments. For these experiments, Cas9 was synthesized and purified using the IPTG induction method with Bacillus edulis containing the Cas9 expression vector pET28b-Cas9-His (#47327, addgene, www.addgene.org / 47327 / ). Each in vitro transcribed gRNA and the purified Cas9 protein were mixed with PCR products containing the target sequences of each gene and cultured at 37°C for 2 hours. After culturing, the reaction was terminated by treatment with an enzyme (proteinase K), and a portion of the reaction mixture was subjected to electrophoresis on a 1% agarose gel to confirm whether or not DNA cleavage had occurred.

[0078] As can be seen from Figure 1, we were able to confirm that the sgRNA-Cas9 complex is active against each target.

[0079] For vector construction of eight of these genes, one target guide RNA sequence corresponding to T1 in Table 1 below was selected for each gene. For vector construction of the remaining four genes, both of the two selected target sequences corresponding to T1 and T2 were used. [Table 1]

[0080] As is clear from Figures 2a and 2b, polycistronic gRNA sequences designed to express target guide RNA sequences of eight genes (SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, and 15) and gRNA sequences of four genes (SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, and 8) were synthesized in rice U6 promoter (GenScript, USA). Then, pSK429 was constructed by substituting the herbicide resistance PPT gene, a selection marker for the pSB11-Cas9 vector (Shim et al., 2018), with the hygromycin resistance gene (HPT).

[0081] Vectors pSK437 and pSK438 were prepared by cleaving pSK429 with HindIII and XbaI and inserting the synthesized polycistronic gRNA sequence. For plant transformation, Agrobacterium tumefaciencs (LBA4404) was transformed with the cloned CRISPR-Cas9 vector.

[0082] Example 2: Plant transformation and sorting Surface-sterilized rice (Oryza sativa) seeds were placed in 2N6 medium (hereinafter referred to as callus induction medium) supplemented with 2N6 salt (Duchefa, Haarlem, Netherlands) and 2 mg / L 2,4-D, and callus formation was induced for 3-4 weeks. Rice transformation was carried out by modifying the method of Hiei et al. (1994). Briefly, the formed callus was cultured at 23°C for 3 days in an Agrobacterium suspension solution (OD600=0.3) transformed with pSK437 and pSK438. After 3 days, the callus was transferred to 2N6CH medium (hereinafter referred to as callus selection medium) supplemented with 40 mg / L hygromycin and 200 mg / L cefotaxime in the callus induction medium. Subsequently, the callus was subcultured in fresh culture medium every two weeks, and the transformed callus was then selected for final production.

[0083] Example 3: Selection of genetically corrected calluses After selecting hygromycin-resistant calluses from 2N6CH medium, whether or not gene transgenicity occurred in these calluses was investigated by PCR using primer sequences targeting surrounding sequences, including the target sequence, as shown in Table 2. [Table 2]

[0084] PCR products were purified using a kit (Expin PCR SV mini kit, GeneAll, South Korea), and then Sanger sequencing was performed to analyze their sequences. INDEL analysis of the nucleotide sequences around the target sequence was performed using an analytical tool (Inference of CRISPR Edits (ICE) analysis tool (ice.synthego.com / # / )), and the callus showing the highest correction efficiency was selected.

[0085] As a result, in obtaining eight gene-mutated cell lines from the group using the pSK437 vector, we selected rice callus line #1-12-20-11, which showed nearly 100% gene correction efficiency in six genes, excluding the β1,3-GalT and α1,4-FucT genes.

[0086] The selection process to obtain four gene-mutant cell lines from the group using the pSK438 vector was carried out in a similar manner.

[0087] Example 4: Acquisition of gene-corrected single cell lines using suspension culture The callus from the #1-12-20-11 line, which showed the highest correction efficiency, was inoculated into 2N6 liquid medium and cultured in suspension at 28°C and 110 rpm. Single cell lines were obtained by subculturing every two weeks.

[0088] The suspension cultures were filtered using a filter (100 μm pore size), and the resulting single cells or single-cell derived cell populations are shown in Figure 3.

[0089] Cells collected by centrifugation were resuspended in 1 mL of 2N6 liquid medium, spread onto 2N6CH agar, and grown until visible to the naked eye. After growing each cell line on 2N6CH agar, they were separated and grown on fresh 2N6CH agar. To obtain single-cell derived cell lines, callus was cleaved from two different sites for each grown cell line callus, dielectric DNA was extracted from it, and PCR was performed around the target sequence for each.

[0090] INDEL analysis was performed using the nucleotide sequences of PCR products from two different sites within a single cell line. Cell lines showing identical INDEL patterns were ultimately evaluated as single-cell derived cell lines (#SC). As a result, two cell line lines were obtained from #SC-26, a cell line with eight gene mutations, and named PMOsC1 and PMOsC2, respectively. To obtain four gene mutation cell lines, two single-cell derived cell line lines were finally selected using a similar method with callus from #19, and named PMOsC3 and PMOsC4, respectively. The gene correction efficiency for each cell line is summarized in Table 3. [Table 3]

[0091] Example 5: Analysis of the structure of N-linked glycans in the acquired cell line. Immunoblotting and liquid chromatography-mass spectrometry (LC-MS) were used to examine the structure of N-linked glycans in the selected cell lines.

[0092] First, for immunoblotting, total protein was extracted from the cell line, and the relative amounts of the plant-specific N-linked glycans α1,3-fucose and β1,2-xylose were compared to wild-type (WT) Dongjin Moemai by Western blotting. Protein was extracted from PBS buffer solution (pH 7.4), and 10 μg of total protein was subjected to PAGE electrophoresis. Immunoblotting was then performed using anti-α1,3-fucose (Agrisera, Sweden) and anti-β1,2-xylose (Agrisera, Sweden) (M, size marker; Lane 1, Dongjin Moemai (WT); lane 2, PMOsC1; lane 3, PMOsC2; lane 4, PMOsC3; lane 5, PMOsC4).

[0093] As a result, as can be seen in Figure 4, it was found that α1,3-fucose and β1,2-xylose were hardly detectable in the selected cell lines. Through these results, we were able to confirm that mutations in the α1,3-FucT and β1,2-XylT enzyme genes occurred on a regular basis. Furthermore, we attempted to analyze the structural changes of N-linked glycans in the selected cell lines in more detail.

[0094] To achieve this, the extracted proteins were treated with trypsin, then treated with PNGase A to separate N-linked glycans from the proteins, and these were analyzed using MALDI-TOF MS (matrix-assisted laser desorption ionization time-of-flight mass spectrometry). [Table 4]

[0095] As can be seen from Table 4, unlike the detection of diverse N-linked glycan structures in the wild (WT) cell line, two types of N-linked glycan structures, MGn and GnGn, were detected in the PMOsC1 and PMOsC2 cell lines, while three types of glycan structures, MM, MGn, and GnGn, were detected in the PMOsC3 and PMOsC4 cell lines. In particular, the proportion of GnGn structures in the PMOsC1 and PMOsC2 cell lines was 80.8% and 92.3%, respectively. On the other hand, in the single PMOsC3 and PMOsC4 cell lines, the MM structure was detected most frequently, but its relative proportion was observed to be 50.8% and 42.3%, respectively. These differences in cell lines are thought to be due to whether or not mutations occurred in the hexosaminidase (HEXA) genes corresponding to Hexo1 to Hexo4. [Table 5] JPEG0007905108000006.jpg44170

[0096] As can be seen in Table 5, the results from secreted proteins secreted into suspension medium by the PMOsC1 cell line showed ratios of 1.0%, 1.6%, and 97.4% for MM, MGn, and GnGn structures, respectively, indicating a more pronounced bias towards GnGn structures than observed in the cells. Through these results, we reconfirmed the mutations of six genes that showed nearly 100% gene correction efficiency in PMOsC1 and PMOsC2, and revealed that the β1,3-GalT and α1,4-FucT genes, which showed relatively low gene correction efficiency, do not actually exhibit a detectable function in protein N-glycosylation.

[0097] The suppression of β1,3-GalT and α1,4-FucT enzyme function in these selected cell lines is likely due to abnormal N-glycosylation resulting from mutations in α1,3-FucT and β1,2-XylT, which act prior to these enzymes. This leads to the creation of abnormal substrate proteins lacking normal N-glycosylation, modified with α1,3-fucose and β1,2-xylose, which are substrates of the β1,3-GalT and α1,4-FucT enzymes.

[0098] In addition, the detection or non-detection of very low levels of the MM structure indicates that the hexosaminidase gene has been mutated.

[0099] Based on the above results, we ultimately obtained PMOsC1 and PMOsC2 single cell lines with mutations in eight genes, and through N-glycosylation structure analysis of these cells, we were able to establish cell lines in which the function of eight target enzymes was suppressed. Furthermore, through analysis of the glycan structure of PMOsC3 and PMOsC4 cell lines, we confirmed that we had successfully induced four gene mutations.

[0100] Such cell lines are expected to be useful resources for establishing efficient cell line systems for conducting environmental stimulus and stress-related experiments in the future, and for studying the function of the relevant genes. Furthermore, these plant-specific glycosylation gene mutant cell lines could be used as production platforms capable of producing various useful proteins.

[0101] Example 6: Expression of trastuzumab, a breast cancer treatment agent, in plant-specific glycosylated mutant cell lines We attempted to express trastuzumab, a breast cancer treatment, in plant-specific glycosylated gene mutant cell lines. After adding the signal peptide sequence (GKHHVTLCCVVFAVLCLASSLAQA) of rice amylase 3E (RAmy3E) protein to the 5' ends of the trastuzumab light and heavy chains, we synthesized trastuzumab light and heavy chain genes optimized for rice codons (GeneArt, Germany) and constructed the pSK446 vector by introducing these genes into a pEAQ-HT vector (Sainsbury et al., 2009). This expression vector was then transformed into PMOsC1 cell lines and selected from 50 mg / L G418 (Geneticin). As a control group, proteins extracted from untransformed WT (Dongjin) callus lines were used. [Table 6] JPEG0007905108000008.jpg90170

[0102] The selected calluses were cultured in suspension for two weeks, and the culture medium was collected. After centrifugation at 440 g for 5 minutes, the medium was concentrated using Vivaspin (50 MWCO, Sartorius). The concentrated solution was immunoblotted using specific antibodies against human IgGγ chains and kappa chains (AP309P, AP502, PMillipore, USA) (1, size marker; 2, concentrated medium used for TMab-transformed cell line suspension culture; 3, WT (negative control); 4, Herzuma (trastuzumab analog, positive control)).

[0103] As can be seen in Figure 5, we confirmed that both light and heavy chains are produced normally in the cell line. [Industrial applicability]

[0104] This invention relates to an N-glycosylation mutant rice (Oryza sativa), a method for producing the same, and a method for producing protein-producing rice using the same. More specifically, it relates to a technology for establishing a rice cell line capable of producing medical proteins by correcting a total of eight genes involved in the plant-specific N-glycosylation process and eliminating plant-specific glycosylation.

Claims

1. A vector for correcting N-glycosylation genes in rice (Oryza sativa) containing a polycistronic guide RNA designed to express the nucleotide sequences of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, and 15, or a polycistronic guide RNA designed to express the nucleotide sequences of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, and 8, and a gene encoding Cas9.

2. The aforementioned guide RNAs are β1,2-XylT (β1,2-xylsiltransferase), α1,3-FucT (α1,3-fucosiltransferase), β1,3-GalT (β1,3-galactosyltransferase), α1,4-FucT (α1,4-fucosiltransferase), HEXO1 (hexosaminidase 1), HEXO2 (hexosaminidase 2), HEXO3 (hexosaminidase 3), and HEXO4 (hexosaminidase) A vector for correcting N-glycosylation genes in rice according to claim 1, which targets the gene in 4) or the genes β1,2-XylT, α1,3-FucT, β1,3-GalT, and α1,4-FucT.

3. N-glycosylated mutant rice (Oryza sativa) transformed with a vector containing a polycistronic guide RNA designed to express the nucleotide sequences of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, and 15, or a polycistronic guide RNA designed to express the nucleotide sequences of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, and 8, and a gene encoding Cas9.

4. The N-glycosylated mutant rice according to claim 3, wherein the N-glycosylated mutant rice is further transformed with a vector containing a gene encoding a target protein.

5. A vector for correcting N-glycosylation genes in rice (Oryza sativa) containing a polycistronic guide RNA designed to express the nucleotide sequences of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, and 15, or a polycistronic guide RNA designed to express the nucleotide sequences of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, and 8, and a gene encoding Cas9; and A trastuzumab expression vector containing a trastuzumab light chain synthesis gene consisting of the nucleotide sequence of SEQ ID NO: 44 and a trastuzumab heavy chain synthesis gene consisting of the nucleotide sequence of SEQ ID NO: 45, which has been codon-optimized to express trastuzumab (TMab) in rice. Rice transformed for trastuzumab production.

6. The guide RNA targets the genes β1,2-XylT, α1,3-FucT, β1,3-GalT, α1,4-FucT, HEXO1, HEXO2, HEXO3, and HEXO4, or targets the genes β1,2-XylT, α1,3-FucT, β1,3-GalT, and α1,4-FucT, according to claim 5, for trastuzumab production.

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