MGAT1-deficient cells and their uses

Abstract

A method for producing modified cells deficient in mannosyl (alpha-1,3-)-glycoprotein beta-1,2-N-acetylglycosaminoltransferase 1 ("MGAT1") activity is provided. Additionally, a CHO cell line deficient in MGAT1 activity produced by the above method is provided. Furthermore, a method for producing a glycoprotein is disclosed.

Description

Technology Field

[0001] Related applications

[0002] This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 502,866 filed May 17, 2023. The entire contents of the aforementioned application are incorporated herein by reference.

[0003] Sequence list

[0004] This invention includes a sequence list that is submitted electronically in .xml format and whose entirety is incorporated herein by reference. A .xml copy created on May 15, 2024, is named "A1000-01200US_20240515_SeqListing.xml" and has a size of 20 kilobytes.

[0005] Field of invention

[0006] The present disclosure relates to a novel cell line and a method for producing a glycoprotein. Background Technology

[0007] The development of broad-spectrum vaccines against pathogenic viruses remains a critical challenge in the fields of immunology and public health. For example, broad-spectrum vaccines are necessary to respond to the continuous emergence of new variants of SARS-CoV-2.

[0008] The major immunogen, the SARS-CoV2 spike protein, is susceptible to mutations and contains a conserved epitope shielded by glycans. Recent studies have revealed that spike protein glycosylation exhibits site-differential effects on viral infectivity, indicating that spike proteins produced by lung epithelial cells upon infection possess a glycoform with higher infectivity. Furthermore, it was found that immunizing mice with spike proteins trimmed to a monoglycosylated state of N-linked glycans elicited a more robust immune response and better protection against SARS-CoV2 variants compared to immunizing with fully glycosylated spike proteins.

[0009] One method of producing viral antigens with altered glycosylation is to mutate the O- or N-glycosylation sites of the antigen. However, this method can alter protein structure, lead to the loss of epitopes, and consequently reduce immunogenicity. Another method involves expressing the viral antigen in cell lines engineered to have an altered glycosylation enzyme pathway.

[0010] Therefore, cell lines and methods for producing glycan-modified glycoprotein antigens that can be used to develop broad-spectrum neutralizing antibodies against viruses and other pathogens are required.

[0011] To meet the above requirements, a method is provided for producing modified cells deficient in mannosyl (alpha-1,3-)-glycoprotein beta-1,2-N-acetylglucosaminyltransferase 1 ("MGAT1") activity. Such a method introduces an RNA-guided endonuclease into a parent cell along with a guide RNA ("gRNA") comprising the sequences described in GGAUGCGCAGACCUGAGCAG (SEQ ID NO: 2), GGUAGUGGAGGACGAUCUGG (SEQ ID NO: 3), UUUCUCCACCUGUAGCAGGG (SEQ ID NO: 4), or GAUCGCCAGGCACUACCGCU (SEQ ID NO: 5); cultures the parent cell; separates a plurality of daughter cells from the cell culture; It includes identifying modified cells deficient in MGAT1 activity.

[0012] The CHO cell line deficient in MGAT1 activity produced by the above method is within the scope of the present invention.

[0013] MGAT1-deficient CHO cells containing nucleic acid having the sequence described in SEQ ID NO: 6 in their genome are also provided.

[0014] A first method for producing a glycoprotein is disclosed by obtaining MGAT1-deficient cells produced using the above method; expressing a protein having an N-glycosylation site in the cells and culturing the cells so that the protein is N-glycosylated by an oligomannose glycan at the N-glycosylation site; and separating the N-glycosylated protein from the cells.

[0015] A second method for producing a glycoprotein is further disclosed by first producing MGAT1-deficient cells using the above method; expressing a protein having an N-glycosylation site in the cells and culturing the cells so that the protein is N-glycosylated by an oligomannose glycan at the N-glycosylation site; and separating the N-glycosylated protein from the cells.

[0016] Finally, a third method for producing a glycoprotein is disclosed, which is performed by obtaining the aforementioned MGAT1-deficient CHO cells containing SEQ ID NO: 6; expressing a protein having an N-glycosylation site in the cells and culturing the cell line so that the protein is N-glycosylated by an oligomannose glycan at the N-glycosylation site; and separating the N-glycosylated protein from the cells.

[0017] Details regarding one or more embodiments of the present invention are described below. Other features, objects, and advantages of the present invention will be apparent from the detailed description, drawings, and claims. Brief explanation of the drawing

[0018] Figure 1 shows Western blot autoradiographs showing SARS-CoV2 spike proteins expressed in three different MGAT1-deficient CHO K1 cell lines that were either untreated (-) or treated (+) with endonuclease H ("EndoH"). Figure 2a shows a Western blot radiograph examining media collected from cultures of MGAT1-deficient CHO K1 cell lines expressing SARA-CoV2 spike proteins ("S protein"), including Sfg, Shm, and Smg, compared to standard proteins. Sfg = fully glycosylated S protein, Shm = gomannose S protein, Smg = monoglycosylated S protein, A = medium from adherent culture, and S = medium from suspension culture. Figure 2b shows a Western blot radiograph examining histidine-tagged S-proteins purified from suspension cultures of MGAT1-deficient CHO K1 cell lines untreated (-) or treated (+) with EndoH, including Shm and Smg, compared to standard proteins. Shm = Gomannose S-protein, Smg = Monoglycosylated S-protein. FIG. 3a is a graph representation of the glycan composition of the 61-F.FSNVT.W fragment of the delta His-tag-free SARS-CoV2 spike protein expressed using exemplary cells according to the present disclosure, treated with or untreated by EndoH. The untreated fragment contains glycans including HexNAc(2)Hex(4), HexNAc(2)Hex(5), HexNAc(2)Hex(6), and HexNAc(2)Hex(7), or does not contain glycans (“un”). The treated fragment contains no glycans or contains only HexNAc(1). Filled squares represent NAc, and filled circles represent mannose. FIG. 3b is a graph of the glycan composition of the 74-F.HAIHVSGTNGTK.R fragment of a delta His-tag-free SARS-CoV2 spike protein expressed using exemplary cells according to the present disclosure, treated with or untreated by EndoH. The untreated fragment contains glycans including HexNAc (1), HexNAc(2)Hex(3), HexNAc(2)Hex(4), and HexNAc(2)Hex(5). The treated fragment contains only HexNAc (1). Filled squares represent NAc, and filled circles represent mannose. FIG. 3c is a graph of the glycan composition of the 122-L.IVNNATNVVIK.V fragment of the delta His-tag-free SARS-CoV2 spike protein expressed using exemplary cells according to the present disclosure, treated with or untreated by EndoH. The untreated fragment contains glycans including HexNAc(2)Hex(4), HexNAc(2)Hex(5), HexNAc(2)Hex(6), HexNAc(2)Hex(7), and HexNAc(2)Hex(8). The treated fragment contains only HexNAc(1). Filled squares represent NAc, and filled circles represent mannose. FIG. 3d is a graph of the glycan composition of the 149-Y.YHKNNKSWMESGVY.S fragment of a delta His-tag-free SARS-CoV2 spike protein expressed using exemplary cells according to the present disclosure, treated with or untreated by EndoH. The untreated fragment contains glycans including HexNAc (2) and Hex (5). The treated fragment contains only HexNAc (1). Filled squares represent NAc, and filled circles represent mannose. FIG. 3e is a graph of the glycan composition of the 163-Y.SSANNCTFEYVSQPF.L fragment of the delta His-tag-free SARS-CoV2 spike protein expressed using exemplary cells according to the present disclosure, treated with or untreated by EndoH. The untreated fragment contains glycans including HexNAc(2)Hex(5) and HexNAc(2)Hex(6). The treated fragment contains only HexNAc(1). Filled squares represent NAc, and filled circles represent mannose. FIG. 3f is a graph representation of the glycan composition of the 232-I.GINIT.R fragment of the delta His-tag-free SARS-CoV2 spike protein expressed using exemplary cells according to the present disclosure, treated with or untreated by EndoH. The untreated fragment contains glycans including HexNAc(1), HexNAc(2)Hex(5), HexNAc(2)Hex(6), HexNAc(2)Hex(7), HexNAc(2)Hex(8), and HexNAc(2)Hex(9), or does not contain glycans (“un”). The treated fragment does not contain glycans or contains only HexNAc(1). Filled squares represent NAc, and filled circles represent mannose. FIG. 3g is a graph of the glycan composition of the 280-K.YNENGTIT.D fragment of the delta His-tag-free SARS-CoV2 spike protein expressed using exemplary cells according to the present disclosure, treated with or untreated by EndoH. The untreated fragment contains glycans including HexNAc(2)Hex(4), HexNAc(2)Hex(5), and HexNAc(2)Hex(6). The treated fragment contains only HexNAc(1). Filled squares represent NAc, and filled circles represent mannose. FIG. 3h is a graph representation of the glycan composition of the 329-V.RFPNIT.N fragment of the delta His-tag-free SARS-CoV2 spike protein expressed using exemplary cells according to the present disclosure, treated with or untreated by EndoH. The untreated fragment contains glycans including HexNAc(1), HexNAc(2)Hex(4), and HexNAc(2)Hex(5), or does not contain glycans (“un”). The treated fragment does not contain glycans or contains only HexNAc(1). Filled squares represent NAc, and filled circles represent mannose. FIG. 3i is a graph representation of the glycan composition of the 341-F.GEVFNAT.R fragment of the delta His-tag-free SARS-CoV2 spike protein expressed using exemplary cells according to the present disclosure, treated with or untreated by EndoH. The untreated fragment contains glycans including HexNAc(1), HexNAc(2)Hex(4), and HexNAc(2)Hex(5). The treated fragment contains mainly HexNAc(1) and a small amount of HexNAc(2)Hex(4). Filled squares represent NAc, and filled circles represent mannose. FIG. 3J is a graph representation of the glycan composition of the 601-S.VITPGTNTS.N fragment of the delta His-tag-free SARS-CoV2 spike protein expressed using exemplary cells according to the present disclosure, treated with or untreated by EndoH. The untreated fragment contains glycans including HexNAc(2)Hex(3), HexNAc(2)Hex(5), and HexNAc(2)Hex(6). The treated fragment contains only HexNAc(1). Filled squares represent NAc, and filled circles represent mannose. FIG. 3k is a graph of the glycan composition of the 614-V.LYQGVNCT.E fragment of the delta His-tag-free SARS-CoV2 spike protein expressed using exemplary cells according to the present disclosure, treated with or untreated by EndoH. The untreated fragment contains glycans including HexNAc(1), HexNAc(2)Hex(5), and HexNAc(2)Hex(6). The treated fragment contains mainly HexNAc(1). Filled squares represent NAc, and filled circles represent mannose. FIG. 3L is a graph representation of the glycan composition of the 655-V.NNSYECDIPI.G fragment of a delta His-tag-free SARS-CoV2 spike protein expressed using exemplary cells according to the present disclosure, treated with or untreated by EndoH. The untreated fragment contains glycans including HexNAc(1), HexNAc(2)Hex(4), and HexNAc(2)Hex(5), or does not contain glycans (“un”). The treated fragment does not contain glycans or contains only HexNAc(1). Filled squares represent NAc, and filled circles represent mannose. FIG. 3m is a graph representation of the glycan composition of the 707-A.YSNNSIA.I fragment of the delta His-tag-free SARS-CoV2 spike protein expressed using exemplary cells according to the present disclosure, treated with or untreated by EndoH. The untreated fragment contains glycans including HexNAc(1), HexNAc(2)Hex(5), HexNAc(2)Hex(6), HexNAc(2)Hex(7), and HexNAc(2)Hex(8), or does not contain glycans (“un”). The treated fragment does not contain glycans or contains only HexNAc(1). Filled squares represent NAc, and filled circles represent mannose. FIG. 3n is a graph representation of the glycan composition of the 715-T.NFTIS.V fragment of the delta His-tag-free SARS-CoV2 spike protein expressed using exemplary cells according to the present disclosure, treated with or untreated by EndoH. The untreated fragment contains glycans including HexNAc(1), HexNAc(2)Hex(4), HexNAc(2)Hex(5), HexNAc(2)Hex(6), HexNAc(2)Hex(7), HexNAc(2)Hex(8), and HexNAc(2)Hex(9), or does not contain glycans (“un”). The treated fragment contains no glycans or contains only HexNAc(1). Filled squares represent NAc, and filled circles represent mannose. FIG. 3o is a graph representation of the glycan composition of the 799-K.TPPIKDFGGFNFS.Q fragment of a delta His-tag-free SARS-CoV2 spike protein expressed using exemplary cells according to the present disclosure, treated with or untreated by EndoH. The untreated fragment contains glycans including HexNAc (1), HexNAc(2)Hex(4), HexNAc(2)Hex(5), HexNAc(2)Hex(6), HexNAc(2)Hex(7), and HexNAc(2)Hex(8). The treated fragment contains only HexNAc (1). Filled squares represent NAc, and filled circles represent mannose. FIG. 3p is a graph representation of the glycan composition of the 1072-A.QEKNFTT.A fragment of the delta His-tag-free SARS-CoV2 spike protein expressed using exemplary cells according to the present disclosure, treated with or untreated by EndoH. The untreated fragment contains glycans including HexNAc (1), HexNAc(2)Hex(4), HexNAc(2)Hex(5), HexNAc(2)Hex(6), and HexNAc(2)Hex(7). The treated fragment contains only HexNAc (1). Filled squares represent NAc, and filled circles represent mannose. FIG. 3q is a graph representation of the glycan composition of the 1096-S.NGTHWFVT.Q fragment of the delta His-tag-free SARS-CoV2 spike protein expressed using exemplary cells according to the present disclosure, treated with or untreated by EndoH. The untreated fragment contains glycans including HexNAc(1), HexNAc(2)Hex(4), HexNAc(2)Hex(5), HexNAc(2)Hex(6), and HexNAc(2)Hex(7), or does not contain glycans (“un”). The treated fragment contains no glycans or contains only HexNAc(1). Filled squares represent NAc, and filled circles represent mannose. FIG. 3r is a graph representation of the glycan composition of the 1132-V.NNTV.Y fragment of the delta His-tag-free SARS-CoV2 spike protein expressed using exemplary cells according to the present disclosure, treated with or untreated by EndoH. The untreated fragment contains glycans including HexNAc (1), HexNAc(2)Hex (4), HexNAc(2)Hex (5), and HexNAc(2)Hex (7). The treated fragment contains only HexNAc (1). Filled squares represent NAc, and filled circles represent mannose. FIG. 3s is a graph representation of the glycan composition of the 1156-K.NHTSPDVDLG.D fragment of the delta His-tag-free SARS-CoV2 spike protein expressed using exemplary cells according to the present disclosure, treated with or untreated by EndoH. The untreated fragment contains glycans including HexNAc(1), HexNAc(2)Hex(4), and HexNAc(2)Hex(5), or does not contain glycans (“un”). The treated fragment does not contain glycans or contains only HexNAc(1). Filled squares represent NAc, and filled circles represent mannose. FIG. 3t is a graph representation of the glycan composition of the 1171-S.GINASVV.N fragment of the delta His-tag-free SARS-CoV2 spike protein expressed using exemplary cells according to the present disclosure, treated with or untreated by EndoH. The untreated fragment contains glycans including HexNAc(1), HexNAc(2)Hex(4), and HexNAc(2)Hex(5), or does not contain glycans (“un”). The treated fragment does not contain glycans or contains only HexNAc(1). Filled squares represent NAc, and filled circles represent mannose. FIG. 3u is a graph representation of the glycan composition of the 655-V.NNSYECDIPI.G fragment of the delta His-tag-free SARS-CoV2 spike protein expressed using exemplary cells according to the present disclosure, treated with or untreated by EndoH. The untreated fragment contains glycans including HexNAc(1), HexNAc(2)Hex(4), and HexNAc(2)Hex(5). The treated fragment contains only HexNAc(1). Filled squares represent NAc, and filled circles represent mannose. Specific details for implementing the invention

[0019] In one embodiment, the present disclosure provides a method for producing a modified cell lacking MGAT1 activity. Such a method comprises introducing, for example, an RNA-guided endonuclease, CHO K1, into a parent cell together with a guide RNA (gRNA).

[0020] As used herein, the terms “MGAT1-deficient” or “MGAT1-inactive deficient” describe the non-function of the mannosyl (alpha-1,3-)-glycoprotein beta-1,2-N-acetylglycosaminoltransferase 1 in cells compared to wild-type or non-modified cells. Defication in MGAT-1 can be achieved by altering the MGAT-1 gene, such as by inserting one or more nucleotides or deleting or substituting one or more nucleotides therein, thereby creating a missense mutation, nonsense mutation, or frame-shift mutation in the gene. The altered gene will no longer be able to express a protein product or will express a non-functional protein product.

[0021] In some embodiments, RNA-guided endonucleases Streptococcus pionogenes Cas9( Streptococcus pyogenes Cas9 is a clustered regularly interspaced short palindromic repeat-associated protein ("Cas"), which may be enhanced SpCas9 (eSpCas9), or SpCas9-high fidelity 1 (SpCas9-HF1). In certain embodiments, the RNA-guided endonuclease is SpCas9.

[0022] Although not intended to be limited to theory, in a specific example where the RNA-guided endonuclease is a Cas protein, the introduced gRNA will bind to the target gene at a specific location based on hybridization between the gRNA and the target gene. Hybridization between the gRNA and the target gene attracts the Cas protein and forms a complex that activates the Cas protein. The activated Cas protein cleaves the binding site and knocks out a fragment of the target gene, leaving a double-strand break (DSB). Subsequently, this DSB will be repaired through a homology-directed repair (HDR) mechanism. Consequently, the repaired gene is no longer intact, having lost its fragment. Such a gene cannot be expressed, or its expressed gene product will lose its function.

[0023] In some embodiments, the gRNA may comprise the nucleic acid sequence described in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. In certain embodiments, the gRNA comprises the nucleic acid sequence described in SEQ ID NO: 3.

[0024] In some embodiments, the parent cell of the present disclosure comprises a gene encoding a protein that exhibits MGAT1 activity. In embodiments in which the gRNA comprises the nucleic acid sequence described in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, the gene of the parent cell encoding the protein that exhibits MGAT1 activity comprises a target sequence that can hybridize with at least a portion of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively.

[0025] In some embodiments, the gRNA used in the method comprises clustered regularly interspaced short palindromic repeat RNA ("crRNA") and trans-activating crRNA ("tracrRNA"). In some embodiments, the crRNA and tracrRNA are coupled to each other through hybridization, whereas in some other embodiments, the crRNA and tracrRNA are formed within the same nucleic acid molecule. In certain embodiments, the tracrRNA comprises the nucleic acid sequence described in SEQ ID NO: 11. It should be noted that in some embodiments, the crRNA comprises the nucleic acid sequence described in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 and further comprises a region complementary to the 5' end of the tracrRNA. For example, the crRNA containing SEQ ID NO: 3 is GGUAGUGGAGGACGAUCUGGGUUUUAGAGCUAUGCU 3' (SEQ ID NO: 13). Exemplary crRNAs containing SEQ ID NOs: 2, 4, and 5 are GGAUGCGCAGACCUGAGCAGGUUUUAGAGCUAUGCU (SEQ ID NO: 12), UUUCUCCACCUGUAGCAGGGGUUUUAGAGCUAUGCU (SEQ ID NO: 14), and GAUCGCCAGGCACUACCGCUGUUUUAGAGCUAUGCU (SEQ ID NO: 15), respectively.

[0026] In some embodiments, RNA-guided endonuclease and gRNA can be introduced into CHO cells, e.g., CHO K1 cells, using electroporation as a pre-formed complex.

[0027] In some embodiments, the method comprises identifying modified cells lacking MGAT1 activity. Such identification may be achieved by using a MGAT1 enzyme activity assay or by sequencing the genomic DNA of candidate cells to identify DNA having insertions and / or deletions that may result in a loss of MGAT1 gene expression and / or a loss of enzyme activity of the MGAT1 gene product. For example, the identification step may be performed by performing a bacteriophage T7 endonuclease I ("T7EI") digestion assay on a genomic DNA fragment amplified from candidate cells and by sequencing the genomic DNA fragment from T7EI digestion assay-positive cells. The T7EI digestion assay is known in the art as an effective tool for identifying small sequence mismatches. For example, a DNA fragment amplified from the genomic DNA of a cell may be denatured and re-annealed with a wild-type DNA molecule. Subsequently, the annealed DNA fragment is digested by T7 endonuclease I, which, if a mismatch exists, produces digested DNA fragments of different sizes. However, the present disclosure is not limited to the use of the T7EI digestion assay.

[0028] In one embodiment of the present disclosure, the modified cells described above may be used to produce a glycoprotein. The first method is characterized by expressing a glycoprotein having an N-glycosylation site in MGAT1-deficient cells (which may be modified CHO cells according to the present disclosure). In some embodiments, such a method is carried out in vitro. The glycoprotein may be expressed by suitable methods in the art, for example, transfection of a protein-expressing vector into modified cells, retroviral transduction, and lentivirus transduction. Such vectors may be, but are not limited to, plasmid expression vectors, retroviral vectors, and lentivirus vectors. Where a viral vector is used, viral particles may be produced in suitable host cells known in the art.

[0029] Expressing a glycoprotein in MGAT1-deficient cells results in N-glycosylation at the N-glycosylation site by oligomannose glycan. Subsequently, the N-glycosylated protein, i.e., the oligomannose glycoprotein, is isolated from the cells by standard procedures known in the art. For example, the N-glycosylated protein can be purified from the culture supernatant. Alternatively, the cells can be collected by centrifugation, and the N-glycosylated protein can be purified from the cell pellet.

[0030] In some embodiments, the culture of MGAT1-deficient cells may be an adherent culture or a suspension culture.

[0031] Additionally, the first method for producing a glycoprotein may include removing the oligomannose glycan to leave only an N-acetylglucosamine residue at the N-glycosylation site, thereby producing a monoglycosylated protein. The oligomannose glycan may be removed by incubating the glycoprotein containing the oligomannose glycan with glycopeptide-D-mannosyl-N4-(N-acetyl-D-glycosaminoyl)2-asparagine 1,4-N-acetyl-beta-glycosaminohydrolase ("Endo H").

[0032] In some embodiments, the glycoprotein expressed in MGAT1-deficient cells may be, for example, a cellular protein or a viral envelope protein.

[0033] In certain embodiments, the glycoprotein is a viral envelope protein, e.g., SARS-CoV2 spike protein, Pan-sarbecovirus spike protein, Pan-betacoronavirus spike protein, or influenza hemagglutinin. The SARS-CoV2 spike protein may be alpha-SARS-CoV2, beta-SARS-CoV2, gamma-SARS-CoV2, delta-SARS-CoV2, omecro-SARS-CoV2, or a variant thereof, but is not limited thereto.

[0034] In certain embodiments, the SARS-CoV2 spike protein may include the amino acid sequence described in SEQ ID NO: 16 or SEQ ID NO: 17.

[0035] In other embodiments, the glycoprotein is a cellular protein, e.g., alpha-fetoprotein, β-human chorionic gonadotropin, cancer antigens 15-3, 19-9, 27.29, 125, and 549, carcinoembryonic antigen, carcinoembryonic antigen-associated cell adhesion molecule, human epidermal growth factor receptor 2, oncofetal fibronectin, placental alkaline phosphatase, and prostate-specific antigen.

[0036] In one embodiment of the present disclosure, two additional methods for producing glycoproteins are provided. In some embodiments, these methods are carried out in vitro.

[0037] A second exemplary method comprises the steps of a first method, except that the second method further comprises generating a modified cell lacking MGAT1 activity.

[0038] A third exemplary method is a special modification of the first method using MGAT1-deficient cells, specifically using MGAT1-deficient cells containing a nucleic acid having the sequence described in SEQ ID NO: 6 in its genome.

[0039] The specific examples provided below are to be interpreted merely as illustrative and not to be interpreted as limiting the remainder of this disclosure in any way. It is expected that a person skilled in the art will be able to make the fullest use of this disclosure based on the description provided herein without further explanation. All publications cited herein are incorporated herein by reference in their entirety.

[0040] Examples

[0041] Example 1: MGAT1 gene editing in CHO cells

[0042] Exemplary adherent CHO K1 cells were cultured in Ham's F12 (Sigma) supplemented with 2 mM GLUTAMAX™ (GIBCO) and 10% fetal bovine serum ("FBS") at 37°C and 5% CO2. For suspension cultures, exemplary CHO K1 cells were also grown in BalanCD CHO Growth A Medium (FUJIFILM Irvine Scientific) supplemented with 8 mM GLUTAMAX™ and 0.5% Anti-Clumping Supplement (FUJIFILM Irvine Scientific) at 37°C and 5% CO2.

[0043] Example 2: Gene Editing

[0044] Gene editing of the MGAT1 gene in CHO K1 cells was performed using standard techniques. In summary, ribonucleoprotein particles ("RNPs") were prepared in vitro by mixing recombinant Cas9, universal tracrRNA (SEQ ID NO: 11), and crRNA containing the sequences described in SEQ ID NOs: 2, 3, 4, or 5. The mixture was incubated at 37°C for 10 minutes to form RNPs.

[0045] RNP (5 microliters) containing 3.3 μM Cas9, 6.6 μM tracrRNA, and one of four crRNAs at 6.6 μM was prepared in 10 μL R buffer using a Neon system (Thermo Fisher) according to manual at a concentration of 1.0 x 10 5 Electroporation was performed into CHO K1 cells. After electroporation, cells were plated in 24-well plates (2 mL per well) and cultured at 37°C and 5% CO2 for 3 days.

[0046] Example 3: Detection of gene-edited clones by T7EI assay

[0047] To examine the results of the gene editing described in Example 2, electroporated cells were prepared at a rate of 1.0 x 10⁻⁶ 5 The cells were grown to reach a certain number, harvested, lysed in 100 μL of lysis buffer (10 mM Tris-HCl pH 8.0, 0.05% SDS, 250 μg / ml Proteinase K) at 37°C for 15 minutes, and heat-inactivated at 85°C for 15 minutes.

[0048] The region of interest of the MGAT1 gene was amplified from a sample of cell lysates by polymerase chain reaction ("PCR") using forward primer ACCCGTGAGGTGTTCCGCCT (SEQ ID NO: 7) and reverse primer AGACACGGGCAAGGAAATCCC (SEQ ID NO: 8) to produce a 943 bp product.

[0049] T7 nuclease E1 digestion assays were performed on PCR products according to the manufacturer's original design (New England Biolabs: "NEB"). T7E1-treated samples were analyzed by agarose gel electrophoresis to identify cells containing insertions / deletions ("indel") in the MGAT1 gene region of interest.

[0050] Indel-positive cell clones were further analyzed by sequencing the 943 bp product after column purification using forward primer ACCCCCTCACCAGCCGTGAT (SEQ ID NO: 9) and reverse primer TCTGGACGAATACAGGCCCGC (SEQ ID NO: 10).

[0051] A cloned cell line was obtained by plating the cells with a series of dilutions.

[0052] Example 4: MGAT1 - Screening of CHO K1 clone cell lines

[0053] The loss of MGAT1 activity in CHO K1 clone cell lines was evaluated by expressing the SARS-CoV2 spike glycoprotein in the cells. Subsequently, the expressed spike protein was incubated with EndoH to detect incompletely glycosylated high-mannose-modified spike proteins. The glycoprotein was expressed in CHO K1 clone cell lines using the lentivirus expression system outlined below.

[0054] Lentivirus generation

[0055] 1.7 x10 7 293T cells (ATCC CRL-3216) were added to 15 cm plates. After 24 hours of incubation, the cells were transfected with a total of 30 μg of four plasmid vectors as follows: 10 μg of a plasmid expressing viral GAG, POL, and ENV genes and containing a REV response element; 2.5 μg of a plasmid expressing REV; 2.5 μg of a plasmid expressing VSV-G protein; and a histidine-tag S protein encoding sequence (wild-type S protein sequence is listed in SEQ ID NO: 16) and a lentivirus backbone. The plasmids were transfected into the 293T cells using Lipofectamine 3000 as directed by the provider (Thermo Fisher). After 48 hours of incubation, the cell supernatant was collected, passed through a 0.45 μm filter, and concentrated 100-fold using a Lenti-X concentrator (Takara) according to the manufacturer's instructions.

[0056] spike protein production

[0057] 30 microliters of concentrated lentivirus 2x10 4 Candidate MGAT1 - It was added to CHO-K1 cloned cells. After one single-cell limiting dilution, 1 x 10⁶ 5 The cells were further amplified until excess cells were reached. Subsequently, 1x10 5 Cells were plated into wells of a 24-well plate containing 0.5 mL of culture medium and grown for 96 hours. The supernatant from each well was collected and purified using COVID-19 spike protein affinity resin (Repligen, SR-24156: NGL#22) according to the manual. Briefly, the collected supernatant was loaded onto a column and traced with 10 mL of Buffer A (20 mM Tris-HCl, 140 mM NaCl, pH 7.5). Subsequently, the column was washed with Buffer A for 8 CVs and eluted with Buffer B (100 mM acetate, 1 M arginine, pH 5.5) for 7 CVs, followed by stripping the column with Buffer C (200 mM acetic acid) for 4 CVs. Afterward, the column was subjected to a cleaning-in-place (CIP) process with Buffer D (0.1 N NaOH) for 4 CVs. Next, the column was neutralized with buffer A for 5 CV. After purification, the collected supernatant was concentrated to a final volume of 20 μL.

[0058] EndoH digestion and Western blot

[0059] Each 10 microliter of concentrated supernatant containing approximately 20 μg of protein was digested with 1 μg EndoH or mock-digested at 37°C for 2 hours as directed by the manufacturer (NEB). MGAT1 -Cells produce gomannose glycoproteins that can be digested by EndoH and exhibit rapid mobility, or gel shift, in acrylamide gel electrophoresis.

[0060] After digestion, 16 μL of each sample was analyzed using Western blot analysis with a BOLT™ system (Thermo Fisher) using an 8% Bis-Tris mini-protein gel as directed by the manufacturer.

[0061] After electrophoretic transfer from the gel to a PVDF membrane, S protein was detected using the primary antibody mouse anti-SARS-CoV2 (clone 1035206; R&D Systems) and the secondary antibody horseradish peroxidase-conjugated goat anti-mouse Fc (Invitrogen), and then visualized using Clarity Max Western ECL substrate (BioRad).

[0062] The results are shown in Figure 1. All three candidate CHO K1 clone cell lines exhibited gel migration toward lower molecular weight S proteins after EndoH digestion, which confirmed the lack of MGAT1 activity in these cells.

[0063] One exemplary CHO K1 clonal cell line was selected for further verification. The MGAT1 gene in these cell lines has a 2-nucleotide deletion at positions 640 and 641 in SEQ ID NO: 1 of the MGAT1 encoding sequence. The 2-nucleotide deletion causes a frameshift in the MGAT1 encoding sequence. The DNA sequence containing the deletion sites is presented in SEQ ID NO: 6.

[0064] Example 5: Optimization of S protein expression

[0065] CHO K1 clonal cell lines were adapted to suspension culture using standard methods. Attachment cultures of the clonal cell lines were grown in 24-well plates, and suspension cultures were grown in T125 flasks. Histidine-tag S protein was expressed in both cell cultures using the method described above.

[0066] The cultured medium was collected (0.5 mL from a well of a 24-well plate and 30 mL from a T125 flask), concentrated to 20 μL, and analyzed using Western blot as described above. The results are shown in Figure 2a.

[0067] Complete glycosylation (S fg ), gomannose glycosylation (S hm ), and monoglycosylation (S mg The standard S protein samples of ) exhibited different mobility as expected. Refer to the first three lanes in Fig. 2.

[0068] Attachment (A) and suspension (S) cultures of the CHO K1 clone cell line expressed significant amounts of S protein after lentivirus infection. Refer to the last two lanes of Fig. 2a. The mobility of S protein was consistent with the gomannose form, indicating that the CHO K1 cell line MGAT1 - It confirmed that it was.

[0069] Histidine-tagged high-mannose S protein was purified from suspension culture medium by nickel chelation using standard techniques, added to EndoH treatment as described in Example 4 above, and analyzed using Western blot. The results are shown in Figure 2b. The untreated S protein purified from the CHO K1 clone cell line moved at the same location, indicating that it is a high-mannose S protein, whereas the EndoH-treated purified S protein moved to a location corresponding to a monoglycosylated S protein.

[0070] Alternatively, His-tag-free SARS-CoV2 spike protein (Delta strain) was expressed by repeating the experiments described in Examples 4 and 5. The expressed and purified S protein was separated into five fractions and treated with chymotrypsin, chymotrypsin and trypsin, trypsin, α-lytic protease and trypsin, and α-lytic protease, respectively. The treated samples were vacuum-dried for MS analysis. The results indicate that the purity of the expressed spike protein samples was over 90%, protease digestion was efficient, and the digestion rate was over 95%.

[0071] After protease digestion, the spike protein was cleaved into 17 peptide fragments containing a total of 21 N-glycosylation sites (peptides containing N17 glycosylation sites were excluded). All 21 N-glycosylation sites, in particular Mannose-5 (Man5), were highly mannosylated (i.e., high mannose content). The high mannose content was further verified using Endo H treatment, as a single GluNAc was observed after treatment (Figs. 3a to 3u).

[0072] Other specific examples

[0073] Example 1. A method for generating modified cells deficient in mannosyl (alpha-1,3-)-glycoprotein beta-1,2-N-acetylglycosaminoltransferase (MGAT1) activity, comprising introducing an RNA-guided endonuclease into a parent cell together with a guide RNA (gRNA) comprising the sequences described in GGAUGCGCAGACCUGAGCAG (SEQ ID NO: 2), GGUAGUGGAGGACGAUCUGG (SEQ ID NO: 3), UUUCUCCACCUGUAGCAGGG (SEQ ID NO: 4), or GAUCGCCAGGCACUACCGCU (SEQ ID NO: 5); culturing the parent cell and amplifying the parent cell; isolating a plurality of daughter cells from the cell culture; identifying modified cells deficient in MGAT1 activity; and isolating said modified cells deficient in MGAT1 activity.

[0074] Example 2. The method of Example 1, wherein identifying modified cells lacking MGAT1 activity comprises performing a bacteriophage T7 endonuclease I (T7EI) digestion assay on genomic DNA fragments amplified from a plurality of daughter cells and sequencing genomic DNA fragments from T7EI digestion assay-positive cells.

[0075] Example 3. The method of Example 1 or Example 2, wherein the gRNA comprises a short palindromic repeat sequence RNA (crRNA) and a trans-activated crRNA (tracrRNA) that appear at regular intervals, and the RNA-guided endonuclease is Cas9.

[0076] Example 4. A method comprising introducing an RNA-guided endonuclease into a cell together with a guide RNA (gRNA) using electroporation of a pre-formed complex of an RNA-guided endonuclease and gRNA in any one of Examples 1 to 3.

[0077] Example 5. A method in any one of Examples 1 to 4, wherein the mother cell is a CHO cell.

[0078] Example 6. The method of Example 5, wherein the gRNA comprises the nucleic acid sequence described in SEQ ID NO: 3.

[0079] Example 7. A Chinese hamster ovary cell line produced according to the method of any one of Examples 1 to 6.

[0080] Example 8. The cell line of Example 7, wherein the cell line is derived from the CHO K1 cell line.

[0081] Example 9. A method for producing a glycoprotein, comprising: obtaining mannosyl (alpha-1,3-)-glycoprotein beta-1,2-N-acetylglycosaminoltransferase-deficient (MGAT1-deficient) cells produced according to the method of any one of Examples 1 to 8; expressing a protein having an N-glycosylation site in the MGAT1-deficient cells so that the protein is N-glycosylated by an oligomannose glycan at the N-glycosylation site; and separating the N-glycosylated protein from the cells.

[0082] Example 10. The method of Example 9, further comprising incubating the glycosylated protein with glycopeptide-D-mannosyl-N4-(N-acetyl-D-glycosaminoyl)2-asparagine 1,4-N-acetyl-beta-glycosaminohydrolase (Endo H) to remove the oligomannose glycan so that the N-acetylglucosamine residue is retained at the N-glycosylated site.

[0083] Example 11. The method of Example 9 or Example 10, wherein the protein is a viral envelope protein.

[0084] Example 12. The method of Example 11, wherein the protein is a viral envelope protein selected from the group consisting of SARS-CoV2 spike protein, Pan-Sarbecovirus spike protein, Pan-Betacoronavirus spike protein, and influenza hemagglutinin.

[0085] Example 13. The method of Example 12, wherein the SARS-CoV2 spike protein is an alpha-SARS-CoV2 spike protein, a beta-SARS-CoV2 spike protein, a gamma-SARS-CoV2 spike protein, a delta-SARS-CoV2 spike protein, or an omichronic-SARS-CoV2 spike protein.

[0086] Example 14. The method of Example 13, wherein the SARS-CoV2 spike protein comprises the amino acid sequence described in SEQ ID NO: 16 or SEQ ID NO: 17.

[0087] Example 15. The method of Example 9 or Example 10, wherein the protein is selected from the group consisting of alpha-fetoprotein, β-human chorionic gonadotropin, cancer antigens 15-3, 19-9, 27.29, 125, and 549, carcinoembryonic antigen, carcinoembryonic antigen-associated cell adhesion molecule, human epidermal growth factor receptor 2, carcinoembryonic fibronectin, placental alkaline phosphatase, and prostate-specific antigen.

[0088] Example 16. A method for producing a glycoprotein, comprising generating mannosyl (alpha-1,3-)-glycoprotein beta-1,2-N-acetylglycosaminoltransferase-deficient (MGAT1-deficient) cells according to the method of any one of Examples 1 to 15; expressing a protein having an N-glycosylation site in the MGAT1-deficient cells so that the protein is N-glycosylated by an oligomannose glycan at the N-glycosylation site; and separating the N-glycosylated protein from the cells.

[0089] Example 17. A Chinese hamster ovary cell line deficient in mannosyl (alpha-1,3-)-glycoprotein beta-1,2-N-acetylglycosaminoltransferase activity, comprising in its genome a nucleic acid having the sequence described in SEQ ID NO: 6.

[0090] Example 18. The cell line of Example 17, wherein the cell line is derived from the CHO K1 cell line.

[0091] Example 19. A method for producing a glycoprotein, comprising obtaining a mannosyl (alpha-1,3-)-glycoprotein beta-1,2-N-acetylglycosaminoltransferase-deficient Chinese hamster ovary cell line of Example 17 or Example 18; expressing a protein having an N-glycosylation site in the cell line so that the protein is N-glycosylated by an oligomannose glycan at the N-glycosylation site; and isolating the N-glycosylated protein from the cell line.

[0092] Example 20. The method of Example 19, further comprising incubating the glycosylated protein with glycopeptide-D-mannosyl-N4-(N-acetyl-D-glycosaminoyl)2-asparagine 1,4-N-acetyl-beta-glycosaminohydrolase (Endo H) to remove the oligomannose glycan, thereby maintaining the N-acetylglucosamine residue at the N-glycosylation site.

[0093] Example 21. The method of Example 19 or Example 20, wherein the protein is a viral envelope protein.

[0094] Example 22. The method of Example 21, wherein the protein is a viral envelope protein selected from the group consisting of SARS-CoV2 spike protein, Pan-Sarbecovirus spike protein, Pan-Betacoronavirus spike protein, and influenza hemagglutinin.

[0095] Example 23. The method of Example 22, wherein the SARS-CoV2 spike protein is an alpha-SARS-CoV2 spike protein, a beta-SARS-CoV2 spike protein, a gamma-SARS-CoV2 spike protein, a delta-SARS-CoV2 spike protein, or an omicron-SARS-CoV2 spike protein.

[0096] Example 24. The method of Example 23, wherein the SARS-CoV2 spike protein comprises the amino acid sequence described in SEQ ID NO: 16 or SEQ ID NO: 17.

[0097] Example 25. The method of Example 19 or Example 20, wherein the protein is selected from the group consisting of alpha-fetoprotein, β-human chorionic gonadotropin, cancer antigens 15-3, 19-9, 27.29, 125, and 549, carcinoembryonic antigen, carcinoembryonic antigen-associated cell adhesion molecule, human epidermal growth factor receptor 2, carcinoembryonic fibronectin, placental alkaline phosphatase, and prostate-specific antigen.

[0098] All features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced by an alternative feature that provides the same, equivalent, or similar purpose. Accordingly, unless otherwise explicitly stated, each disclosed feature is merely an example of a general series of equivalent or similar features.

[0099] From the foregoing description, a person skilled in the art can easily grasp the essential features of the present disclosure and can modify and alter the invention to suit various uses and conditions without departing from the spirit and scope of the present disclosure. Accordingly, other embodiments are also within the scope of the claims.

[0100] Sequence list SEQ ID NO: 01 atgctgaaga agcagtctgc agggcttgtg ctttggggtg ctatcctctt tgtgggctgg aatgccctgc tgctcctctt cttctggaca cgcccagccc ctggcaggcc cccctcagat agtgctatcg atgatgaccc tgccagcctc acccgtgagg tgttccgcct ggctgaggac gctgaggtgg agttggagcg gcagcgggg ctgttgcagc aaatcaggga gcatcatgct ttgtggagac agaggtggaa agtgcccacc gtggcccctc cagcctggcc ccgtgtgcct gcgaccccct caccagccgt gatccccatc ctggtcattg cctgtgaccg cagcactgtc cggcgctgct tggataagtt gttgcactat cggccctcag ctgagcattt ccccatcatt gtcagccagg actgcgggca cgaagaca gcacaggtca ttgcttccta tggcagtgca gtcacaca tccggcagcc agacctgagt aacatcgctg tgcccccaga ccaccgcaag ttccagggtt actacaagat cgccaggcac taccgctggg cactgggcca gatcttcaac aagttcaagt tcccagcagc tgtggtagtg gaggacgatc tggaggtggc accagacttc tttgagtact tccaggccac ctacccactg ctgagaacag acccctccct ttggtgtgtg tctgcttgga atgacaatgg caaggagcag atggtagact caagcaaacc tgagctgctc tatcgaacag acttttttcc tggccttggc tggctgctga tggctgagct gtggacagag ctggagccca agtggcccaa ggccttctgg gatgactgga tgcgcagacc tgagcagcgg aaggggcgggcctgtattcg tccagaaatt tcaagaacga tgacctttgg ccgtaagggt gtgagccatg ggcagttctt tgatcagcat cttaagttca tcaagctgaa ccagcagttc gtgtctttca cccagttgga tttgtcatac ttgcagcggg aggcttatga ccgggatttc cttgcccgtg tctatagtgc ccccctgcta caggtggaga aagtgaggac caatgatcag aaagtgaggac caatgatcag aaaggtctgg gggaggtgcg ggtacagtac actagcagag acagcttcaa ggcctttgct aaggccctgg gtgtcatgga tgacctcaag tctggtgtcc ccagagctgg ctaccggggc gttgtcactt tccagttcag gggtcgacgt gtccacctgg cacccccaca aacctgggaa ggctatgatc ctagctggaa ttag SEQ ID NO: 02 ggaugcgcag accugagcag SEQ ID NO: 03 gguaguggag gacgaucugg SEQ ID NO: 04 uuucuccacc uguagcaggg SEQ ID NO: 05 gaucgccagg cacuaccgcu SEQ ID NO: 06 agttcaagtt cccagcagct gtggtagtgg aggacgatgg aggtggcacc agacttcttt gagt SEQ ID NO: 07 acccgtgagg tgttccgcct SEQ ID NO: 08 agacacgggc aaggaaatcc c SEQ ID NO: 09 accccctcac cagccgtgat SEQ ID NO: 10 tctggacgaa tacaggcccg c SEQ ID NO: 11 agcauagcaa guuaaaauaa ggcuaguccg uuaucaacuu gaaaaaagugg caccgagucg gygcuuu SEQ ID NO: 12 ggaugcgcag accugagcag guuuuagagc uaugcu SEQ ID NO: 13 gguaguggag gacgaucugg guuuuagagc uaugcu SEQ ID NO: 14 uuucuccacc uguagcaggg guuuuagagc uaugcu SEQ ID NO: 15 gaucgccagg cacuaccgcu guuuuagagc uaugcu SEQ ID NO: Spike protein of 16WT strain (Wuhan strain) MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT SEQ ID NO: Spike protein of strain 17delta MFVFLVLLPL VSSQCVNLRT RTQLPPAYTN SFTRGVYYPD KVFRSSVLHS TQDLFLPFFSNVTWFHAIHV SGTNGTKRFD NPVLPFNDGV YFASIEKSNI IRGWIFGTTL DSKTQSLLIVNNATNVVIKV CEFQFCNDPF LDVYYHKNNK SWMESGVYSS ANNCTFEYVS QPFLMDLEGKQGNFKNLREF VFKNIDGYFK IYSKHTPINL VRDLPQGFSA LEPLVDLPIG INITRFQTLLALHRSYLTPG DSSSGWTAGA AAYYVGYLQP RTFLLKYNEN GTITDAVDCA LDPLSETKCTLKSFTVEKGI YQTSNFRVQP TESIVRFPNI TNLCPFGEVF NATRFASVYA WNRKRISNCVADYSVLYNSA SFSTFKCYGV SPTKLNDLCF TNVYADSFVI RGDEVRQIAP GQTGKIADYNYKLPDDFTGC VIAWNSNNLD SKVGGNYNYR YRLFRKSNLK PFERDISTEI YQAGSKPCNGVEGFNCYFPL QSYGFQPTNG VGYQPYRVVV LSFELLHAPA TVCGPKKSTN LVKNKCVNFNFNGLTGTGVL TESNKKFLPF QQFGRDIADT TDAVRDPQTL EILDITPCSF GGVSVITPGTNTSNQVAVLY QGVNCTEVPV AIHADQLTPT WRVYSTGSNV FQTRAGCLIG AEHVNNSYECDIPIGAGICA SYQTQTNSRG SAGSVASQSI IAYTMSLGAE NSVAYSNNSI AIPTNFTISVTTEILPVSMT KTSVDCTMYI CGDSTECSNL LLQYGSFCTQ LNRALTGIAV EQDKNTQEVFAQVKQIYKTP PIKDFGGFNF SQILPDPSKP SKRSFIEDLL FNKVTLADAG FIKQYGDCLGDIAARDLICA QKFNGLTVLP PLLTDEMIAQ YTSALLAGTI TSGWTFGAGA ALQIPFAMQMAYRFNGIGVT QNVLYENQKLIANQFNSAIG KIQDSLSSTA SALGKLQNVV NQNAQALNTLVKQLSSNFGA ISSVLNDILS RLDPPEAEVQ IDRLITGRLQ SLQTYVTQQL IRAAEIRASANLAATKMSEC VLGQSKRVDF CGKGYHLMSF PQSAPHGVVF LHVTYVPAQE KNFTTAPAICHDGKAHFPRE GVFVSNGTHW FVTQRNFYEP QIITTDNTFV SGNCDVVIGI VNNTVYDPLQPELDSFKEEL DKYFKNHTSP DVDLGDISGI NASVVNIQKE IDRLNEVAKN LNESLIDLQELGKYEQYIKW PLVPRGSPGS GYIPEAPRDG QAYVRKDGEW VLLSTFLG

Claims

Claim 1 A method for producing a glycoprotein in vitro, wherein the method comprises obtaining mannosyl (alpha-1,3-)-glycoprotein beta-1,2-N-acetylglycosaminoltransferase-deficient (MGAT1-deficient) cells; expressing a protein having an N-glycosylation site in MGAT1-deficient cells so that the protein is N-glycosylated by an oligomannose glycan at the N-glycosylation site; A method produced by a method comprising isolating an N-glycosylated protein from an MGAT1-deficient cell, wherein the MGAT1-deficient cell contains SEQ ID NO: 6 in its genome, and the MGAT1-deficient cell introduces an RNA-guided endonuclease into a parent cell along with a guide RNA (gRNA) containing a sequence described in GGAUGCGCAGACCUGAGCAG (SEQ ID NO: 2), GGUAGUGGAGGACGAUCUGG (SEQ ID NO: 3), UUUCUCCACCUGUAGCAGGG (SEQ ID NO: 4), or GAUCGCCAGGCACUACCGCU (SEQ ID NO: 5); culturing the parent cell and amplifying the parent cell; isolating a plurality of daughter cells from the cell culture; identifying a modified cell lacking MGAT1 activity; and isolating the modified cell lacking MGAT1 activity. Claim 2 A method according to claim 1, further comprising incubating an N-glycosylated protein with glycopeptide-D-mannosyl-N4-(N-acetyl-D-glycosaminoyl)2-asparagine 1,4-N-acetyl-beta-glycosaminohydrolase (Endo H) to remove oligomannose glycans so that N-acetylglucosamine residues are retained at the N-glycosylated site. Claim 3 A method according to claim 1, wherein the protein is a viral envelope protein. Claim 4 A method according to claim 3, wherein the viral envelope protein is selected from the group consisting of SARS-CoV2 spike protein, Pan-sarbecovirus spike protein, Pan-betacoronavirus spike protein, and influenza hemagglutinin. Claim 5 In claim 4, the SARS-CoV2 spike protein is an alpha-SARS-CoV2 spike protein, a beta-SARS-CoV2 spike protein, a gamma-SARS-CoV2 spike protein, a delta-SARS-CoV2 spike protein, or an omichron-SARS-CoV2 spike protein. Claim 6 In claim 5, the SARS-CoV2 spike protein comprises SEQ ID NO: 16 or SEQ ID NO:

17. Claim 7 The method of claim 1, wherein the protein is selected from the group consisting of alpha-fetoprotein, β-human chorionic gonadotropin, cancer antigens 15-3, 19-9, 27.29, 125, and 549, carcinoembryonic antigen, carcinoembryonic antigen-related cell adhesion molecule, human epidermal growth factor receptor 2, oncofetal fibronectin, placental alkaline phosphatase, and prostate-specific antigen. Claim 8 A method for producing a glycoprotein in vitro, comprising obtaining a mannosyl (alpha-1,3-)-glycoprotein beta-1,2-N-acetylglycosaminoltransferase-deficient Chinese hamster ovary cell line, wherein the mannosyl (alpha-1,3-)-glycoprotein beta-1,2-N-acetylglycosaminoltransferase-deficient Chinese hamster ovary cell line having SEQ ID NO: 6 in its genome; expressing a protein having an N-glycosylation site in the cell line so that the protein is N-glycosylated by an oligomannose glycan at the N-glycosylation site; and isolating the N-glycosylated protein from the cell line. Claim 9 A method according to claim 8, further comprising incubating an N-glycosylated protein with glycopeptide-D-mannosyl-N4-(N-acetyl-D-glycosaminoyl)2-asparagine 1,4-N-acetyl-beta-glycosaminohydrolase (Endo H) to remove oligomannose glycans so that N-acetylglucosamine residues are retained at the N-glycosylated site. Claim 10 In paragraph 8, a method in which the protein is a viral envelope protein. Claim 11 A method according to claim 10, wherein the viral envelope protein is selected from the group consisting of SARS-CoV2 spike protein, Pan-Sarbecovirus spike protein, Pan-Betacoronavirus spike protein, and influenza hemagglutinin. Claim 12 In claim 11, the SARS-CoV2 spike protein is an alpha-SARS-CoV2 spike protein, a beta-SARS-CoV2 spike protein, a gamma-SARS-CoV2 spike protein, a delta-SARS-CoV2 spike protein, or an omicro-SARS-CoV2 spike protein. Claim 13 In claim 12, the SARS-CoV2 spike protein comprises SEQ ID NO: 16 or SEQ ID NO:

17. Claim 14 A method according to claim 8, wherein the protein is selected from the group consisting of alpha-fetoprotein, β-human chorionic gonadotropin, cancer antigens 15-3, 19-9, 27.29, 125, and 549, carcinoembryonic antigen, carcinoembryonic antigen-associated cell adhesion molecule, human epidermal growth factor receptor 2, carcinoembryonic fibronectin, placental alkaline phosphatase, and prostate-specific antigen. Claim 15 delete Claim 16 delete Claim 17 delete Claim 18 delete Claim 19 delete Claim 20 delete Claim 21 delete Claim 22 delete Claim 23 delete Claim 24 delete Claim 25 delete

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