Targeted integrated cell and application thereof

Abstract

The invention provides a cell genome site capable of being used for targeted integration of exogenous nucleic acid molecules. The site is located in a nucleic acid sequence corresponding to a cell genome NC048595.1: 454997734-455024715 of a Chinese hamster ovary CHO-K1. The cell genome sites can integrate foreign protein genes and perform stable expression. The invention also provides stable and high-yield targeted integrated cells, such as host cells derived from Chinese Hamster Ovary (CHO), methods for their production and uses thereof.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and genetic engineering, specifically involving specific sites in the cellular genome and their application in constructing targeted integration cells. Background Technology

[0002] In the biopharmaceutical field, Chinese hamster ovary (CHO) cells are the mainstream host cell for producing recombinant protein drugs (such as monoclonal antibody drugs). In traditional cell line development, the target gene mainly enters the host genome through random integration. Due to the genomic "position effect," exogenous target genes are highly likely to insert into heterochromatin regions or transcriptionally silent regions, resulting in low and unstable protein expression levels. Furthermore, the clonal heterogeneity caused by random integration makes the process of screening high-yield and stable-yield cell lines highly unpredictable, often requiring months of large-scale screening, and making it difficult to ensure batch-to-batch consistency in the production process.

[0003] To overcome the limitations of random integration, site-specific integration (SSI) technology has emerged. SSI involves integrating a transgene encoding a target recombinant protein into a pre-defined genomic site, providing an effective way to produce clones with higher uniformity and shorten cell line development cycles. SSI has become a promising strategy for developing exogenous expression host cells (such as CHO cell lines), allowing research teams to repeatedly target preferred genomic sites that have been proven to have high activity and stable expression characteristics.

[0004] Currently, various technologies, such as Cre / LoxP, Flp / FRT recombinase systems, and CRISPR / Cas9 gene editing technology, have been used to achieve site-specific integration. Furthermore, multiple sites have been reported in this field that can be used to integrate exogenous target genes to stably express target proteins. Compared to random integration, site-specific integration using highly active genomic sites significantly improves the efficiency and accuracy of integration.

[0005] However, providing more universal, efficient, and long-term stable highly active genomic sites for site-specific integration remains a core task in this field. Summary of the Invention

[0006] To address the aforementioned technical problems, one objective of this invention is to provide a cellular genomic locus. This cellular genomic locus allows for the insertion of exogenous genes, supports high-level transcription of the exogenous genes, maintains the genetic stability of the exogenous genes during cell passage, and ensures that normal cell function is not disrupted and cell phenotype and growth stability are maintained after the exogenous gene is integrated into the locus.

[0007] Another object of the present invention is to provide the application of the said cellular genomic site for targeted integration of exogenous nucleic acid molecules.

[0008] Another object of the present invention is to provide nucleic acid molecules for gene-targeted integration based on the site and recombinant cells obtained by integration, as well as the use of said nucleic acid molecules and recombinant cells in the production of recombinant proteins, particularly antibody drugs.

[0009] The technical solution of the present invention is as follows.

[0010] First aspect

[0011] This invention provides a cellular genomic locus that can be used for targeted integration of exogenous nucleic acid molecules. The locus is located in the nucleic acid sequence of the corresponding Chinese hamster ovary CHO-K1 cell genome NC_048595.1: 454997734-455024715.

[0012] Further, the cell genomic locus is preferably located in the nucleic acid sequence corresponding to the nucleotide sequence shown in SEQ ID NO: 1; the nucleotide sequence shown in SEQ ID NO: 1 is CHO cell genome NC_048595.1: 455009789-455017197. More preferably, the cell genomic locus is located in the nucleic acid sequence corresponding to the nucleotide sequence shown in SEQ ID NO: 2; the nucleotide sequence shown in SEQ ID NO: 2 is CHO cell genome NC_048595.1: 455012801-455016320. More preferably, the cell genomic locus is located in the nucleic acid sequence corresponding to the nucleotide sequence shown in SEQ ID NO: 3; the nucleotide sequence shown in SEQ ID NO: 3 is CHO cell genome NC_048595.1: 455014081-455014880. Particularly preferably, the cell genomic site is located in the nucleic acid sequence corresponding to the nucleotide sequence shown in SEQ ID NO: 4; the nucleotide sequence shown in SEQ ID NO: 4 is CHO cell genome NC_048595.1:455014170-455014757.

[0013] Furthermore, the nucleic acid sequence can be a nucleic acid sequence contained in the genome of a eukaryotic cell. The eukaryotic cell can be a mammalian cell, preferably a Chinese hamster ovary (CHO) cell, including but not limited to CHO-K1, CHO-S, CHO-DG44, CHO-DXB11 (DUXB11), CHO-GS knockout cell lines (such as CHO-K1SV GS-KO), and ExpiCHO cell lines; human embryonic kidney cells, including but not limited to HEK293, HEK293T, HEK293F, HEK293E, and Expi293; human retinal cells (such as PER.C6); human amniotic fluid cells (such as CAP-T); mouse myeloma cells, such as NSO and Sp2 / 0; African green monkey kidney cells (Vero); and young hamster kidney cells (BHK).

[0014] Second aspect

[0015] This invention provides the application of the cellular genomic sites described in the first aspect in the targeted integration of exogenous nucleic acid molecules, wherein the application involves the targeted integration of exogenous nucleic acid molecules into the cellular genomic sites. This targeted integration can be achieved through homology-directed repair (HDR).

[0016] The exogenous nucleic acid molecule can be a DNA molecule, such as a single-stranded DNA molecule or a double-stranded DNA molecule, used to express one or more exogenous proteins. Furthermore, the application described in this aspect further includes the expression of one or more exogenous proteins in cells.

[0017] According to a specific embodiment of the present invention, the exogenous nucleic acid molecule may include: a first recombination recognition sequence and a second recombination recognition sequence recognized by a recombinase, and one or more expression cassettes located between the first recombination recognition sequence and the second recombination recognition sequence, wherein the expression cassette includes the exogenous nucleic acid sequence and a promoter driving its expression.

[0018] The first and second recombination recognition sequences can be generated by a recombinase. Optionally, the recombinase can be a tyrosine recombinase or a serine recombinase. For example, the tyrosine recombinase is Cre recombinase or FLP recombinase; the serine recombinase is Bxb1 integrase or ΦC31 integrase.

[0019] Accordingly, the first recombination recognition sequence and the second recombination recognition sequence may contain sites that can be recognized by tyrosine recombinases (e.g., FRT sites or Lox sites, such as LoxP, Lox71, Lox66, etc.), or sites that can be recognized by serine recombinases (e.g., att sites, such as attB, attP, attL, attR, etc.).

[0020] According to a specific embodiment of the present invention, the first recombination identification sequence and the second recombination identification sequence may independently include the following sequences: LoxP sequence, LoxPL3 sequence, LoxP 2L sequence, LoxFas sequence, Lox511 sequence, Lox2272 sequence, Lox2372 sequence, Lox5171 sequence, Loxm2 sequence, Lox71 sequence, Lox66 sequence, FRT sequence, Bxb1 attP sequence, Bxb1 attB sequence, attP sequence, attB sequence, attL sequence, and attR sequence.

[0021] Preferably, the first recombination identification sequence and the second recombination identification sequence may independently include the following sequences: LoxP sequence, LoxPL3 sequence, LoxP 2L sequence, LoxFas sequence, Lox511 sequence, Lox2272 sequence, Lox2372 sequence, Lox5171 sequence, Loxm2 sequence, Lox71 sequence, and Lox66 sequence. For example, the first recombination identification sequence may include the lox71 sequence, and the second recombination identification site may include the lox2272 sequence; or vice versa.

[0022] The exogenous nucleic acid sequence may be a nucleic acid sequence or gene encoding a target protein (POI), a selection marker, and / or a reporter protein.

[0023] For example, the exogenous nucleic acid sequence may be a nucleic acid sequence or gene encoding one or more proteins selected from: antibodies (such as the heavy and / or light chains of immunoglobulins or any fragment thereof, single-chain antibodies, bispecific antibodies, nanobodies), antigen-binding fragments, fusion proteins, cytokines, growth factors, hormones, coagulation factors, enzymes, receptor proteins (such as chimeric antigen receptors CAR, T-cell receptors TCR), structural proteins or functional fragments thereof.

[0024] For example, the exogenous nucleic acid sequence may be a nucleic acid sequence or gene encoding a selection marker and / or a reporter protein, such as antibiotic resistance genes (e.g., resistance genes against genimycin, puromycin, neomycin, hygromycin, bleomycin), metabolic selection marker genes (e.g., glutamine synthase GS gene, dihydrofolate reductase DHFR gene, thymidine kinase gene), and fluorescent protein or luminescent protein genes (e.g., GFP, RFP, luciferase gene).

[0025] Depending on the purpose, the exogenous nucleic acid sequence may be any one or more of the above-mentioned nucleic acid sequences or genes.

[0026] According to a specific embodiment of the present invention, the exogenous nucleic acid sequence is a nucleic acid sequence or gene encoding a selection marker and / or a reporter protein, that is, the expression cassette may contain a nucleic acid sequence or gene encoding a selection marker and / or a reporter protein, as well as an optional promoter driving its expression.

[0027] According to a specific embodiment of the present invention, the exogenous nucleic acid sequence is a nucleic acid sequence or gene encoding a target protein (POI), that is, the expression cassette may contain a nucleic acid sequence or gene encoding a target protein (POI) and an optional promoter to drive its expression.

[0028] The expression cassette may contain one or more promoters that drive the expression of the exogenous nucleic acid sequence. The promoter may be selected from one or more of the following: CMV promoter, SV40 promoter, RSV promoter, β-globin promoter, UBC promoter, EF1a promoter, ubiquitin promoter, β-actin promoter, PGK1 promoter, Rosa26 promoter, HSP70 promoter, GAPDH promoter, Eif4A1 promoter, Egr1 promoter, FerH promoter, SM22α promoter, or Endothelin-1 promoter.

[0029] Furthermore, the exogenous nucleic acid molecule can be a vector, such as a lentiviral vector, adenovirus vector, adeno-associated virus vector, herpesvirus vector, poxvirus vector, baculovirus vector, papillomavirus vector, papillomavirus vector, integrative phage vector, non-viral vector, transposon and / or transposase, integrase substrate, or plasmid.

[0030] Preferably, the nucleic acid molecule is a donor vector or a targeting plasmid. More preferably, the nucleic acid molecule is a homologous recombination donor plasmid or a donor plasmid for recombinase-mediated cassette exchange; the plasmid may be supercoiled circular DNA, relaxed circular DNA, or linearized DNA.

[0031] Furthermore, the exogenous nucleic acid molecule may also include a 5' homology arm (HA) and a 3' homology arm, thereby having a structure from the 5' to the 3' end: [5' homology arm]-[first recombination recognition sequence]-[one or more expression cassettes]-[second recombination recognition sequence]-[3' homology arm].

[0032] The 5' and 3' homologous arms each contain nucleotide sequences homologous to the nucleic acid sequences surrounding the cellular genomic sites described in the first aspect of the invention.

[0033] Further, the 5' homologous arm and the 3' homologous arm each comprise a nucleotide sequence of 15-10000 bases in length. Preferably, the 5' homologous arm and the 3' homologous arm each comprise a nucleotide sequence of 100-5000 bases in length, more preferably 300-4000 bases in length, more preferably 500-3000 bases in length, further preferably 600-2000 bases in length, and particularly preferably 800-1500 bases in length.

[0034] Further, the 5' homologous arm comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher, or even 100%, sequence identity with the nucleotide sequence shown in SEQ ID NO: 13; and / or, the 3' homologous arm comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher, or even 100%, sequence identity with the nucleotide sequence shown in SEQ ID NO: 14.

[0035] Third aspect

[0036] The present invention provides a nucleic acid molecule comprising: a first recombinant recognition sequence and a second recombinant recognition sequence recognized by a recombinase, and one or more expression cassettes located between the first recombinant recognition sequence and the second recombinant recognition sequence, the expression cassettes comprising a foreign nucleic acid sequence and a promoter driving its expression.

[0037] The nucleic acid molecule can be a DNA molecule, such as a single-stranded DNA molecule or a double-stranded DNA molecule.

[0038] The first and second recombination recognition sequences can be generated by a recombinase. Optionally, the recombinase can be a tyrosine recombinase or a serine recombinase. For example, the tyrosine recombinase is Cre recombinase or FLP recombinase; the serine recombinase is Bxb1 integrase or ΦC31 integrase.

[0039] Accordingly, the first recombination recognition sequence and the second recombination recognition sequence may contain sites that can be recognized by tyrosine recombinases (e.g., FRT sites or Lox sites, such as LoxP, Lox71, Lox66, etc.), or sites that can be recognized by serine recombinases (e.g., att sites, such as attB, attP, attL, attR, etc.).

[0040] According to a specific embodiment of the present invention, the first recombination identification sequence and the second recombination identification sequence may independently include the following sequences: LoxP sequence, LoxPL3 sequence, LoxP 2L sequence, LoxFas sequence, Lox511 sequence, Lox2272 sequence, Lox2372 sequence, Lox5171 sequence, Loxm2 sequence, Lox71 sequence, Lox66 sequence, FRT sequence, Bxb1 attP sequence, Bxb1 attB sequence, attP sequence, attB sequence, attL sequence, and attR sequence.

[0041] Preferably, the first recombination identification sequence and the second recombination identification sequence may independently include the following sequences: LoxP sequence, LoxPL3 sequence, LoxP 2L sequence, LoxFas sequence, Lox511 sequence, Lox2272 sequence, Lox2372 sequence, Lox5171 sequence, Loxm2 sequence, Lox71 sequence, and Lox66 sequence. For example, the first recombination identification sequence may include the lox71 sequence, and the second recombination identification site may include the lox2272 sequence; or vice versa.

[0042] The exogenous nucleic acid sequence may be a nucleic acid sequence or gene encoding a target protein (POI), a selection marker, and / or a reporter protein.

[0043] For example, the exogenous nucleic acid sequence may be a nucleic acid sequence or gene encoding one or more proteins selected from: antibodies (such as the heavy and / or light chains of immunoglobulins or any fragment thereof, single-chain antibodies, bispecific antibodies, nanobodies), antigen-binding fragments, fusion proteins, cytokines, growth factors, hormones, coagulation factors, enzymes, receptor proteins (such as chimeric antigen receptors CAR, T-cell receptors TCR), structural proteins or functional fragments thereof.

[0044] For example, the exogenous nucleic acid sequence may be a nucleic acid sequence or gene encoding a selection marker and / or a reporter protein, such as antibiotic resistance genes (e.g., resistance genes against genimycin, puromycin, neomycin, hygromycin, bleomycin), metabolic selection marker genes (e.g., glutamine synthase GS gene, dihydrofolate reductase DHFR gene, thymidine kinase gene), and fluorescent protein or luminescent protein genes (e.g., GFP, RFP, luciferase gene).

[0045] Depending on the purpose, the exogenous nucleic acid sequence may be any one or more of the above-mentioned nucleic acid sequences or genes.

[0046] According to a specific embodiment of the present invention, the exogenous nucleic acid sequence is a nucleic acid sequence encoding a selection marker and / or a reporter protein, that is, the expression cassette may contain a nucleic acid sequence or gene encoding a selection marker and / or a reporter protein, as well as an optional promoter driving its expression.

[0047] According to a specific embodiment of the present invention, the exogenous nucleic acid sequence is a nucleic acid sequence or gene encoding a target protein (POI), that is, the expression cassette may contain a nucleic acid sequence or gene encoding a target protein (POI) and an optional promoter to drive its expression.

[0048] The expression cassette may contain one or more promoters that drive the expression of the transgene, and the promoter may be selected from one or more of the following: CMV promoter, SV40 promoter, RSV promoter, β-globin promoter, UBC promoter, EF1a promoter, ubiquitin promoter, β-actin promoter, PGK1 promoter, Rosa26 promoter, HSP70 promoter, GAPDH promoter, Eif4A1 promoter, Egr1 promoter, FerH promoter, SM22α promoter, or Endothelin-1 promoter.

[0049] The nucleic acid molecule may be a recombinant DNA vector, such as a lentiviral vector, adenovirus vector, adeno-associated virus vector, herpesvirus vector, poxvirus vector, baculovirus vector, papillomavirus vector, papillomavirus vector, integrative phage vector, non-viral vector, transposon and / or transposase, integrase substrate, or plasmid.

[0050] Preferably, the nucleic acid molecule is a donor vector or a targeting plasmid. More preferably, the nucleic acid molecule is a homologous recombination donor plasmid or a donor plasmid for recombinase-mediated cassette exchange; the plasmid may be supercoiled circular DNA, relaxed circular DNA, or linearized DNA.

[0051] Furthermore, the nucleic acid molecule also includes a 5' homologous arm and a 3' homologous arm, thereby having a structure from the 5' to the 3' end: [5' homologous arm] - [first recombination recognition sequence] - [one or more expression cassettes] - [second recombination recognition sequence] - [3' homologous arm].

[0052] The 5' and 3' homologous arms each contain nucleotide sequences homologous to the nucleotide sequences surrounding the cellular genomic sites described in the first aspect of the invention.

[0053] Further, the 5' homologous arm and the 3' homologous arm each comprise a nucleotide sequence of 15-10000 bases in length. Preferably, the 5' homologous arm and the 3' homologous arm each comprise a nucleotide sequence of 100-5000 bases in length, more preferably 300-4000 bases in length, more preferably 500-3000 bases in length, further preferably 600-2000 bases in length, and particularly preferably 800-1500 bases in length.

[0054] Further, the 5' homologous arm comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher, or even 100%, sequence identity with the nucleotide sequence shown in SEQ ID NO: 13; and / or, the 3' homologous arm comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher, or even 100%, sequence identity with the nucleotide sequence shown in SEQ ID NO: 14.

[0055] Fourth aspect

[0056] The present invention also provides a targeted integration cell comprising the nucleic acid molecule described in the third aspect of the present invention. The targeted integration cell can be obtained by targeted integration of the nucleic acid molecule into a host cell. The targeted integration can be achieved through homology-directed repair (HDR) and optionally recombinase-mediated cassette exchange (RMCE).

[0057] The host cell can be a eukaryotic cell. The eukaryotic cell can be a mammalian cell, preferably a Chinese hamster ovary (CHO) cell, including but not limited to CHO-K1, CHO-S, CHO-DG44, CHO-DXB11 (DUXB11), CHO-GS knockout cell lines (such as CHO-K1SV GS-KO), and ExpiCHO cell lines; human embryonic kidney cells, including but not limited to HEK293, HEK293T, HEK293F, HEK293E, and Expi293; human retinal cells (such as PER.C6); human amniotic fluid cells (such as CAP-T); mouse myeloma cells, such as NSO and Sp2 / 0; African green monkey kidney cells (Vero); and young hamster kidney cells (BHK).

[0058] Preferably, the host cell is a Chinese hamster ovary (CHO) cell, such as CHO-K1, CHO-S, CHO-DG44, CHO-DXB11 (DUXB11), CHO-GS knockout cell lines (such as CHO-K1SV GS-KO), and ExpiCHO cell lines; more preferably, the host cell is a CHO-K1 cell.

[0059] Fifth aspect

[0060] The present invention also provides a method for preparing targeted integrated cells as described in the fourth aspect, the method comprising: targeting and integrating the nucleic acid molecules into the host cells via homology-directed repair (HDR).

[0061] Furthermore, the homology-directed repair can be mediated by the CRISPR / Cas9 system.

[0062] In the CRISPR / Cas9 system, the sgRNA sequence can target the nucleotide sequence shown in SEQ ID NO: 3.

[0063] Preferably, the CRISPR / Cas9 system contains the following sgRNA sequence:

[0064] GGACAGGCATTTCCTACGGG (SEQ ID NO: 5);

[0065] TGGGACAGGCATTTCCTACG (SEQ ID NO: 6);

[0066] CATCGAAGTCCAAGTTTTAT (SEQ ID NO: 7).

[0067] According to a specific embodiment of the present invention, the preparation method includes the following steps: introducing the nucleic acid molecule, the sgRNA and the Cas enzyme into the host cell to induce genetic damage and achieve homologous targeted repair using the nucleic acid molecule as a template; and then screening to obtain targeted integration cells.

[0068] In the above steps, preferably, the nucleic acid molecule and the co-expression plasmid of the sgRNA and Cas enzyme are introduced into the host cell. More preferably, the nucleic acid molecule and the co-expression plasmid of the sgRNA and Cas enzyme are introduced into the host cell by electrotransfection.

[0069] Optionally, the preparation method further includes the following step: replacing the exogenous nucleic acid sequence of the targeted integrated nucleic acid molecule in the obtained cell with other target genes via recombinase-mediated cassette exchange (RMCE). For example, if the nucleic acid molecule contains a selectable marker gene and a reporter gene, the selectable marker gene and the reporter gene are replaced with antibody heavy chain and / or light chain encoding genes.

[0070] Sixth aspect

[0071] The present invention also provides a protein production method, the method comprising: culturing the targeted integration cells described in the fourth aspect of the present invention, and collecting the protein expressed by the nucleic acid molecules.

[0072] Compared with the prior art, the inventors of this invention discovered a genomic site in the genome of Chinese hamster ovary (CHO) cells. Extensive experiments have demonstrated that this site allows the insertion of exogenous nucleic acid sequences, supports high-level transcription of exogenous nucleic acid sequences, maintains the genetic stability of exogenous nucleic acid sequences during cell passage, and when exogenous nucleic acid sequences are integrated into this site, the normal function of the cell is not disrupted, and the phenotype and growth stability of the cell can be maintained.

[0073] Based on the above findings, this invention provides the application of the genomic site in the targeted integration of exogenous nucleic acid molecules and a targeted integration cell, which is constructed by targeted integration of exogenous nucleic acid molecules into the specific site. Experiments have demonstrated that this targeted integration cell has the advantages of high expression of exogenous nucleic acid molecules and stable passage. Furthermore, due to its robust and high-yield characteristics, the cell has low requirements for subsequent culture processes (e.g., culture conditions, culture medium formulation), exhibiting good process adaptability. Simultaneously, because the exogenous nucleic acid molecule insertion site provided by this invention is determined, compared to traditional random integration cell construction methods, the construction process required for the targeted integration cell of this invention is simpler, significantly shortening development time. While improving efficiency and reducing costs, it also greatly reduces the uncertainty of the development process, demonstrating excellent reproducibility and controllability.

[0074] Furthermore, the targeted integration cell line provided by this invention can serve as a platform cell, laying the foundation for the subsequent development of recombinant protein expression cells. For example, when the purpose is antibody expression or production, the recombinase recognition site contained in the targeted integration cell line provided by this invention can serve as a "landing pad," used to efficiently and precisely replace the integrated exogenous nucleic acid molecules with antibody light and heavy chain coding genes through recombinase-mediated cassette exchange (RMCE) technology, thereby rapidly constructing antibody expression cell lines. Attached Figure Description

[0075] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0076] Figure 1 : Plasmid map of the donor repair plasmid in Example 1.

[0077] Figure 2 : Statistical graph of EGFP expression level in targeted integration cell line in Example 1.

[0078] Figure 3 : Stability verification diagram of the targeted integration cell line in continuous passage in Example 1.

[0079] Figure 4 The plasmid map used to express the target product in Example 2 of this invention.

[0080] Figure 5 : Statistical chart of antibody expression levels in targeted integration cell lines after RMCE in Example 2 of this invention. Detailed Implementation

[0081] To make the objectives, technical solutions, embodiments, and beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings.

[0082] It should be understood that the specific embodiments and examples described herein are merely illustrative of the invention and are not intended to limit the scope of protection of the invention in any way. The fundamental purpose of providing these embodiments is to enable those skilled in the art to more thoroughly understand the contents disclosed in this application.

[0083] This invention can be implemented in various different forms and should not be limited to the embodiments described herein. Any modifications, equivalent substitutions, or improvements made by those skilled in the art based on the core ideas disclosed in this application without departing from the spirit and scope of this invention should be considered to fall within the protection scope of this application. Furthermore, for ease of understanding, the following description may contain numerous specific details; however, those skilled in the art should understand that the implementation of this invention does not necessarily depend on all of these specific details.

[0084] Unless the context otherwise requires, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is intended only to describe particular embodiments and is not intended to limit the scope of the invention.

[0085] Unless otherwise specified, the ordinal numbers such as "first" and "second" mentioned in the context of this invention are used only to distinguish different technical features and do not represent any limitation on order, relative importance or quantity.

[0086] In the context of this invention, any numerical range mentioned should be understood to include its two endpoints and encompass all consecutive values ​​within that range and all subranges. For example, the numerical range “1 to 10” includes 1 and 10 and all values ​​in between; if the range refers to integers, it includes 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0087] In the context of this invention, the use of "about" or "approximately" to modify numerical values ​​indicates that the value is within an acceptable range of error that can be determined by a person skilled in the art, which depends in part on the method of measurement or the limitations of the system. For example, "about" or "approximately" may refer to a range of ±10%, preferably ±5%, more preferably ±1% of the given value being modified; in biological systems or processes, "about" or "approximately" may also refer to a range of one order of magnitude, five times, or two times.

[0088] In the context of this invention, percentages (%) are used unless otherwise specified, where % (w / w) or wt% refers to weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0089] In the context of this invention, the term "antibody" is used in its broadest sense, encompassing a variety of structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (such as bispecific antibodies), and antibody fragments, provided they retain the desired antigen-binding activity. In the context of this invention, the term "antibody fragment" refers to a molecule containing a portion of the antigen-binding region of an antibody, such as Fv, Fab, Fab', F(ab')2, scFv, biantibodies, and linear antibodies.

[0090] In the context of this invention, the term "targeted integration cell" is used interchangeably with the term "site-specific integration," both referring to cells in which exogenous nucleic acid molecules, exogenous nucleotide sequences, or exogenous genes have been introduced. This term also includes the progeny of such cells, regardless of the number of passages. It should be understood that the nucleic acid content of progeny cells may not be entirely identical to that of the parent cells due to mutations, but as long as they retain the same function or biological activity as the initially transformed cells, they are covered by this term.

[0091] In the context of this invention, the term "vector" (or "recombinant vector") refers to a nucleic acid tool capable of carrying and amplifying another nucleic acid molecule linked to it into a host cell. A vector can be a self-replicating, independent structure, such as a plasmid, or a sequence integrated into the host cell's genome. If the vector contains regulatory elements (such as a promoter) that drive the expression of the linked nucleic acid, it is called an "expression vector."

[0092] In the context of this invention, the term "homologous" (or "homologous sequence") refers to nucleic acid or protein sequences that show significant sequence similarity after alignment. The similarity or identity between two sequences can be approximately 70%, 80%, 90%, 95%, 99%, or higher. Alignment can be performed using standard algorithms and procedures such as BLAST and FASTA.

[0093] In the context of this invention, the term "integration site" refers to a specific nucleic acid location in the cell genome for the insertion of a foreign nucleotide sequence. This location can be between two adjacent nucleotides or at any point in a nucleotide sequence.

[0094] Compared to existing technologies, this invention identifies a highly stable, site-specific insertion or targeted integration site within the cell genome capable of expressing exogenous nucleic acid molecules at specific locations. This site may be located in the genome of Chinese hamster ovary CHO-K1 cells NC_048595.1: 454997734-455024715; further, it is located in the following sequences:

[0095] SEQ ID NO: 1:

[0096]

[0097] SEQ ID NO: 2:

[0098]

[0099] SEQ ID NO: 3:

[0100] GCCACAGAGAAACCCTGTCTCGAAAAATAAAATAAAATAAATAAATAAATACAGTTTTAGGTGCAAAGCAGATAAAGTGGCTGGGGTTGCCCGCCCCCCCCCCCCCCGTAGGAAATGCCTGTCCCAGATTAAAATCTTAGTCAGTGCCCCAGTGAGAGGCTAAGAGGGTCAGGTATATGTCCCAGCCTGCTAACCCTTATTCCAGCTAGGTCTGTGGTTCAGGCCTCCAGATCCACAGTCATCTGCTCTGCAATGGGTTGATGTGAGCTCCAGGTCCCAGCCACAAGTTTCCATGACTACCCACCCTTCCTGCTTTAGGCTGAGCTTCCACCCAAAGGATGCTTCTTTCTTTCTTGGTTCTCTGTGTTTTATCAAGCCCATGCCCTGCTGGAAGCATTCCCCATATCTAGAAAGATCTTTTTTATCATCCTACTTATTCCTCAACCTGGTGGTGTTCCTGCCTCTTTCTGCCCAGGGACTCCAATGCTCCAAGTTCTGTTGTCATCATGACAGAGAGGGGGTTCAGGTCTCTTCAGGGGGACAAGTGTGACAAGACTTTTCCTGGGGAGATGCAACCTAAGCCTACTCACCCCAGACAGGGAGCCCATGACAGACCAAAGTAGGACACCATCGAAGTCCAAGTTTTATTGGGATTACTTACAAGAATGGGGTGAGGGCTACTTAGAGTAGCAGAATGACTCATAGACAGCTGCATCACCACAATCTGAAGAGTCCTAGTGTAGACAGACCAGCTGAGCTGCCTGAGTCACTGGAAGTGGTACTTCCAGCCTGTTGAACTG

[0101] SEQ ID NO: 4:

[0102] CCCGCCCCCCCCCCCCCGTAGGAAATGCCTGTCCCAGATTAAAATCTTAGTCAGTGCCCCAGTGAGAGGCTAAGAGGGTCAGGTATATGTCCCAGCCTGCTAACCCTTATTCCAGCTAGGTCTGTGGTTCAGGCCTCCAGATCCAC AGTCATCTGCTCTGCAATGGGTTGATGTGAGCTCCAGGTCCCAGCCACAAGTTTCCATGACTACCCACCCTTCCTGCTTTAGGCTGAGCTTCCACCCAAAGGATGCTTCTTTCTTTCTTGGTTCTCTGTGTTTTATCAAGCCCATGC CCTGCTGGAAGCATTCCCCATATCTAGAAAGATCTTTTTTATCATCCTACTTATTCCTCAACCTGGTGGTGTTCCTGCCTCTTTCTGCCCAGGGACTCCAATGCTCCAAGTTCTGTTGTCATGACAGAGAGGGGGTTCAGGTCT CTTCAGGGGGACAAGTGTGACAAGACTTTTCCTGGGGAGATGCAACCTAAGCCTACTCACCCCAGACAGGGAGCCATGACAGACCAAAGTAGGACACCATCGAAGTCCAAGTTTTATTGGGATTACTTACAAGAATGGGGTGAGGG

[0103] The present invention will be described below with reference to specific embodiments. Those skilled in the art will understand that these embodiments are for illustrative purposes only and do not limit the scope of the invention in any way.

[0104] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available products.

[0105] Example 1 Establishment of host cell lines for targeted integration

[0106] This embodiment provides a process for establishing a CHO host cell line with site-directed integration. Its technical principle is based on the CRISPR / Cas9-mediated homology-directed repair (HDR) mechanism.

[0107] Specifically, by co-transfecting CHO cells with sgRNA, Cas9 expression plasmid, and donor repair plasmid, the Cas9 protein, guided by sgRNA, precisely cleaves the genomic target NC_048595.1 (455014081-455014880) shown in SEQ ID NO: 3, generating a double-strand break (DSB). Subsequently, the cell's HDR repair mechanism is activated, and using the donor repair plasmid as a repair template, the exogenous nucleotide sequence cassette located between the homologous arms (HAs) carried on the donor repair plasmid, which are homologous to the sequences flanking the genomic break site, is precisely inserted into the genomic site.

[0108] In this embodiment, the non-coding region between the Ndufv2 and Washc1 genes shown in SEQ ID NO: 2 was selected as the integration "safe harbor" (named "N2W1"), aiming to maintain the normal function of endogenous genes (e.g., protein translation) and thus preserve the cell's phenotype and growth stability. The inserted exogenous nucleotide sequence cassette includes a fluorescent reporter gene (EGFP), an resistance selection gene (PuroR), and two recombinase recognition sites (lox sites). The resulting fluorescently labeled host cell lines can be identified and screened using flow cytometry (FACS) and polymerase chain reaction (PCR).

[0109] In this embodiment, the insertion of the exogenous nucleotide sequence cassette into N2W1 as described above is compared with the insertion of the same exogenous nucleotide sequence cassette into the Fer1L4 target gene (see patent publication WO2013190032).

[0110] The specific process is as follows.

[0111] 1. Plasmid construction

[0112] First, construct the following two plasmids:

[0113] a) Co-expression plasmid of sgRNA and Cas9:

[0114] Commercially available plasmids were used to construct sgRNA and Cas9 co-expression plasmids targeting N2W1 and sgRNA and Cas9 co-expression plasmids targeting Fer1L4 (control).

[0115] The sgRNA sequence targeting N2W1 is as follows:

[0116] (1) GGACAGGCATTTCCTACGGG (SEQ ID NO: 5);

[0117] (2) TGGGACAGGCATTTCCTACG (SEQ ID NO: 6);

[0118] (3) CATCGAAGTCCAAGTTTTAT (SEQ ID NO: 7).

[0119] The sgRNA sequence that recognizes the Fer1L4 target gene is as follows:

[0120] (1) ACAGGGATTGATCCAGTGCA (SEQ ID NO: 8);

[0121] (2) CACCTCTCCAGTGAGCACCT (SEQ ID NO: 9);

[0122] (3) AGTCTCATGTTGGTATAAGC (SEQ ID NO: 10).

[0123] b) Donor Plasmid: See schematic diagram Figure 1 .

[0124] like Figure 1 As shown, the donor repair plasmid contains two homologous arms that target transcription hotspots. Between the two homologous arms is an expression cassette flanked by recombinase recognition site 1 (lox71) and recombinase recognition site 2 (lox2272). Inside the expression cassette is a fluorescent reporter gene (EGFP) driven by a promoter and a eukaryotic resistance selection gene (PuroR) expressed in tandem with a 2A peptide.

[0125] Commercially available plasmids were used to construct and synthesize donor repair plasmids targeting N2W1 and Fer1L4 (control).

[0126] The recombinase recognition sites 1 and 2 are identical in both plasmids, and their sequences are as follows:

[0127] Sequence of the Lox71 recognition site:

[0128] ATAACTTCGTATAATGTATGCTATACGAACGGTA (SEQ ID NO: 11);

[0129] Sequence of the Lox2272 recognition site:

[0130] ATAACTTCGTATAAAGTATCCTATACGAAGTTAT (SEQ ID NO: 12).

[0131] The inserted homologous arm sequences of the present invention in N2W1 are as follows:

[0132] N2W1-5’ homologous arm:

[0133] CAGAGGTCCTGAGTTCAATTCTCGGCAATCACATGGTGGCTCACAACCATCTTGCATGAGATCTGGTGCCCCCTGCTGGCGTGCAGGCAGAAGACTGTATACATAATAAATAAATAAAATCTTTTAAAAATTCAAAAAATATAGACTGTTCAGGGAAAGAGTAAAGCACACTAAAAGTATGGGGTGTGTTTCTCAAAGGAGAGTTGCACTTCATTGTGTATTCTGTGGCCAAACATACAGTTTTGGTGAATTCTGAAATTATTTTCAAATGGTTGCTATGTAATTTAAATGAGGGAACTAGAGAGTTGGTTCAGTGGTTAAGAGCACTGGTTGCTCTAGCAGAGGATAGTAGTTACATTCTCTGCACCTACAGGAGAGCTCACAGCCATCCTTAGCTCCAGTTCCAGGGGACCTGATGCCCTCTTCTGGCCTCTCCAGGCACCAGGCAGGCAAGTAATATACAAACATGTATGCAGGTATAACACCCATACACGTAAAATTTTAAAATAATTTTTCAAAGGGCTCCCTTTGTTTTTGTTTTTAAAGAGTAAAAGACCTCGGGTTAAGCCTGACTCTAGACTGAACTCCAAAGTCAGGAAAACTACTGGTAGAACTGAGTTCAACGGCAGGTGGTTGGTGCCACACGCTTTTAACGTCAGCACTCGGGAGGCAGAGGCAGGCAGATCTCTGTGAGTTCAAGACCAGCCTGGTCTACAAGAATTAGTTCCAGGACAGCCTCCAAAGCCACAGAGAAACCCTGTCTCGAAAAATAAAATAAAATAAATAAATAAATACAGTTTTAGGTGCAAAGCAGATAAAGTGGCTGGGGTTGCCCGCCCCCCCCCCCCCC (SEQ ID NO: 13)

[0134] N2W1-3’ homologous arm:

[0135] TATTGGGATTACTTACAAGAATGGGGTGAGGGCTACTTAGAGTAGCAGAATGACTCATAGACAGCTGCATCACCACAATCTGAAGAGTCCTAGTGTAGACAGACCAGCTGAGCTGCCTGAGTCACTGGAAGTGGTACTTCCAGCCTGTTGAACTGCAGGCCGGCTGTGCAGTGTGCTCCCGGCTCCCAGAGCACAAATAAGTGTGAGCTGTCACTGTGCTGGGGTAGGTTTTGGCAAATTTGTATTGCTTTCACATCATGATAAACTTAAAAGGCTGTAAACCAAAGCCCTGGAAGTCTGGGATTGTAACATAAGGACGAGCTCTTCTACTACAGGCCAGCTGGCTCAGGCTTTGCCATTGACGGAGAAGGAGCGCCTCCTGGTGGAGAAGAGGAGCTCAGGCCTAGAGGGAGATTTAATCTTGCATGGACTGGACAGTTTTAATACCCAGAACTTTTGTGTGGTGATATTTTGTTTGTAATCTAATAAAGCTTGCCTGAAGATCAGAATGCAAAGCTGAGTCCTAGTTAGCCACACACCTAGTTAAGAGCTAAGCCACGAGCTAATCACACACACACACACACACACACACACACACACACACACACACACACACACACAGAGAGCAAGCCACTAATTCGCTGCACACCTAAGCCACTACTTAGCTACAGAGGACAGGCAGTGGTGACCACACCTTTTATTCCAGAACTCGGGAGACAGAGACAGATGGATCTCTGTGAGTTCCAGGCCAGCCTGGGTTACATGAGAGTGAGTCAGTCTAAAAAAGAAACAGAGCTCACATTTTTGATCCCAGCACTTGGGATGACACCACACAAGTCATGGAAACTTG (SEQ ID NO: 14)

[0136] The inserted homologous arm sequence on the Fer1L4 gene is as follows:

[0137] Fer1L4 - 5' homologous arm:

[0138] CTGGCCTAAACTCATGGAATAACTTCCCTACTTCCAGGAGAGCAGTCTGGGGACCCCTTCGTGGCTAAAGCAGGACAATCCAGCCGGCTCCTGAAGGTGATGGGGTGTTCCTGAGGCTGGAGAGGGGGATGTCAGGGTTTGTGGTGGTGCTGACTGAGAGCAACAAGGTCCTCTCTGCCTCAGGCTCCTCTGAAGAAGCTCACCTTAGGGCTCCTGGGTCAAGGTCCTGAGCTGGAGGAGGACATCCCAGACCCAGAAGAGATGGACTGGTGGTCCAAGTACTATGCCTCACTGCAGGAGCTCCAGGGGCAGGTGGGTGGAGCTCCAGGGGCAGGTGGATGGAGCTCCAGGGGCAGGTGGATGGAGCTCCAGGGGCAGGTGGGCACAGCACAACCTAGGGCAGGAACTGGGGGAGGAAAGGCAAGAAGGTGGGGAGTCTCATCAAACAAGACCACTGTGTGTCCCAGAGCTCTTTATTGCGGACATCTTTTCTCCTCTATACCACAATTTTAGTAGTTGGACATTAGTACTTCTGCCTCCCAACACTCTGTTGCTCTAATAAAATAGAACATGAGGGGCTGGAGAGATGGCTCAGAGGTTAAGAGCACTGGCTGTTCTTCCAGAGTTTCTGAGTTCAATTCCCAGCAACCACATAGTGGTTCACAGCCATCTATAATGAGATCTGGTGCTCTCTTCTGGTGTGCAGGCATACATGCAGGCAGAACATTGTATACATAATAAACAATAAATCCTTAAAAAAAAAAAAAAAGAACATGAGGCCAGCATGGTGGTGCACAGCCTTAATCCCTGCACTGGATCAATCCCTGTGAGTTCGAGGCCAGCCTGGTCTATATAACAGGCAGTGAGACCTGTCTCTGAAACAAAAAACATGAGTGTAGAATTTTAGCTCTTAAACTTGGGTATGGTGACACACACCTCTCCAGTGAGCA (SEQ ID NO: 15)

[0139] Fer1L4 - 3' homologous arm:

[0140] TATACCAACATGAGACTCTGTCTCAAATTTTTTTTATTTTAATTTTTGTTAATAGCCACATTTAAAAAGTGAAAGGGAACAAATGAGAATAATTTTTGGTACTGTTTTATTAATGTATACAAAATATTGTTCCAACCAGTGATTATCATAAAAATTAGTAATATGCTATTTTACATTCTCACAGTAAGTCTGCTGTGTGTCCCACTCACACCACCTCACAGTGCAGACTGACCTCACTTCAAGGGCTCAGTGACCGTGGGTGGGTGGTGGTGACTGAAGTCAACAGCATAGGTTTAGACAATAGAGGGGACTTTAAAAAGAAGTGACTGTGGCTGAAGAAGAGGGGTGGGCCAAGGGGGGCAGTGGGAGGGGCTACCTCCGACCTGTCTTTTGATTTCAGTCCGGCTTTGATGAGGATGAGATGGAGGACGCTGGGGACCCAGGTGAGAGTCCTGACTTTTGTTCTTGCTCCATCTCTCCACCCATCCCTGTCCCCACATTCTTACCTGGACCATTTCTGCTGTTTCCAGACGGGACCCACCTCATTTCTGGGGACAGGGAGGCTCAGGAGCAGGGAAGAACTGATATCAAAGTGTCTGGACCTCAGAAGAAAGCAATCGCCACCTTGAAGGTGACAAGGTGTTGGGGAGAAGGGTGCCCCTTGGATTTGGGGAACAGAGTTGTCACTGAGACCCCTCACCTTTCAGATCTACAACAGCTCCCTAGAGGAGGAGTTTAACCACTTTGAGGACTGGCTGAATGTGTTCCCCCTGTACAGAGGGCAAGGCGGCCAGGCTGGAGATGGCGAGGAAGGTTCTGGACACTTGGTGGGCAAATTCAAGGTATACTTGGGGGAAGAATGGGAGACTGACAAGGGACA (SEQ ID NO: 16)

[0141] The two plasmids were linearized using PvuI restriction endonuclease, and the linear DNA was purified and recovered.

[0142] 2. Cell transfection (site-specific integration construction)

[0143] Take 2 × 10⁶ CHO-K1 cells in logarithmic growth phase 7 Each sample was centrifuged and resuspended in an appropriate amount of electroporation buffer. This example establishes two parallel experiments:

[0144] Experimental group (T2507): The constructed sgRNA / Cas9 co-expression plasmid targeting the site in SEQ ID NO: 3 was mixed with the linearized donor repair plasmid (total mass 20 μg) at a mass ratio of 1:1;

[0145] Control group (Fer1L4): The constructed sgRNA / Cas9 co-expression plasmid targeting the Fer1L4 site was mixed with the linearized donor repair plasmid (total mass 20 μg) at a mass ratio of 1:1.

[0146] The above plasmid mixture was co-transfected into CHO-K1 cells.

[0147] 3. Screening and identification of stable cell pools

[0148] Immediately after electroporation, cells were transferred to basal medium and incubated overnight at 37°C and 5% CO2 to recover. After 24 hours, the medium was replaced with pressure selection medium 1 containing puromycin for pressure selection. After approximately one week of culture, both groups yielded fluorescent cell pools with stable resistance.

[0149] The obtained cell pools were analyzed using flow cytometry. The results showed that the average fluorescence intensity of the experimental group (T2507) cell pools was comparable to that of the control group (Fer1L4) cell pools (see [link]). Figure 2 This indicates that the site-specific integration efficiency and expression level of the site in SEQ ID NO: 3 are comparable to those of the validated Fer1L4 site.

[0150] 4. Screening and stability evaluation of monoclonal cell lines

[0151] Pool cells from the experimental and control groups, which were in good condition, were subjected to limiting dilutions and cultured in cloning medium to isolate monoclonal cells. After 14 days, fluorescent monoclonal cells were selected and transferred to basal medium for expansion culture. Subsequently, cells were cultured in fed-batch culture mode using fed-batch medium, during which cell growth curves and fluorescence intensity were monitored. Based on growth and expression data, monoclonal cell lines with good growth curves and high fluorescence intensity were selected and named: T2507-54 (fluorescent protein site-specific integration at the gene site in SEQ ID NO: 3, more specifically in SEQ ID NO: 4); and Fer1L4-02 (fluorescent protein site-specific integration at the Fer1L4 gene site, as a positive control).

[0152] 5. Study on passaging stability

[0153] T2507-54 and Fer1L4-02 cell lines were passaged consecutively in basal medium to a doubling time of 60 passages (PDL60), and fluorescence stability was tested. Results are as follows: Figure 3 As shown, the T2507-54 cell line exhibited minimal fluctuations in fluorescence intensity and stable cell growth during long-term passage, demonstrating high comparability in stability and expression levels compared to the control cell line Fer1L4-02. These results confirm that the site in SEQ ID NO: 3 is suitable for constructing a high-yield, stable cell line.

[0154] Example 2 Establishment of antibody expression cell lines

[0155] In this embodiment, the lox site contained in the T2507-54 cell line established in Example 1 was used as the "landing pad." A target exchange plasmid containing the antibody light and heavy chain coding genes was used, and recombinase-mediated cassette exchange was employed to replace the EGFP reporter gene and the resistance selection gene PuroR with the antibody light and heavy chain coding genes, thus establishing an antibody expression cell line. The specific process is as follows.

[0156] 1. Construction of the target exchange plasmid: See schematic diagram. Figure 4 .

[0157] like Figure 4 As shown, the target exchange plasmid contains antibody B heavy light chain expression cassettes flanking recombinase recognition site 1 (lox66) and recombinase recognition site 2 (lox2272) in the same direction.

[0158] A target exchange vector (containing the MSX resistance gene) was constructed using commercially available plasmids; this vector will also serve as a control vector for random integration.

[0159] The recombinase recognition site 1 (lox66) sequence contained in the plasmid is as follows:

[0160] Lox66 recognition sites:

[0161] TACCGTTCGTATAATGTATGCTATACGAAGTTAT (SEQ ID NO: 17)

[0162] The target exchange plasmid was linearized using the PvuI enzyme, and the DNA was purified and recovered.

[0163] 2. Construction of Gene Exchange (RMCE) and Control Group

[0164] This experiment established an experimental group (fixed-point integration) and a control group (randomized integration), and the specific operation is as follows:

[0165] Experimental group (T2507-RMCE): T2507-54 cells (logarithmic growth phase, total amount 2×10⁻⁶) constructed in Example 1 were used. 7 The DNA was resuspended in electroporation buffer. 15 μg of linearized antibody expression exchange plasmid and 5 μg of Cre enzyme expression plasmid were mixed at a mass ratio of 3:1 (total DNA 20 μg) and co-transfected into cells.

[0166] Control group (Random): Wild-type CHO-K1 cells (logarithmic growth phase, total amount 2×10⁻⁶) were collected. 7 Each cell was resuspended in electroporation buffer. 20 μg of linearized antibody expression exchange plasmid (without Cre enzyme plasmid) was transfected into cells to construct a randomized integration control.

[0167] 3. Screening and Monoclonalization:

[0168] Twenty-four hours after transfection, the two groups of cells were seeded into bulkpools. Subsequently, pressure selection was performed using pressure selection medium 2 containing 25 μM MSX.

[0169] Obtaining a Bulkpool: After culturing for approximately 7-10 days, once the cells have resumed growth and their viability is stable, a stable cell pool (Bulkpool) is obtained. Cells in the experimental group that have undergone correct exchange exhibit loss of fluorescence and MSX resistance. These are designated as T2507-Bulkpool (site-specific integration pool) and Random-Bulkpool (random integration pool), respectively.

[0170] Obtaining monoclonal clones: The two types of Bulkpool cells were cultured in a large-scale manner, and then monoclonalized using the limiting dilution method. After screening and identification (non-fluorescent clones were screened in the experimental group), monoclonal cell lines with high expression were obtained and designated as T2507-54-11 (site-specific integration monoclonal clone) and Random-10 (random integration monoclonal clone), respectively.

[0171] Once the cell lines have expanded to the shake-flask stage and the cell viability, growth rate, and density have returned to normal, they will be uniformly fed-batch culture for evaluation.

[0172] 4. Fed-batch culture and expression level assessment:

[0173] The four cell lines mentioned above (two pool groups and two single clone groups) were respectively treated with 1×10⁴ cells / mL. 6 Cells were seeded at a density of 100 cells / mL in 125mL shake flasks (inoculation volume 25mL) and cultured using fed-batch medium. All cell lines followed the same feeding strategy: fed-batch medium was added according to the plan on days 4, 7, 9, and 11 (addition amounts of 4%, 4%, 5%, and 4%, respectively); glucose was added on days 3, 5, 7, 9, 11, and 13 to maintain glucose levels based on cellular glucose consumption.

[0174] Detection point: Collect samples on day 14 of culture and detect antibody expression levels.

[0175] Figure 5 The final antibody yield of each cell line on day 14 is shown. The results indicate that, at the pool level, the antibody expression level of the site-specific integration cell pool (T2507-Bulkpool) using the method of this invention is significantly better than that of the random integration control pool (Random-Bulkpool); while at the monoclonal level, the expression level of the site-specific integration monoclonal cell line (T2507-54-11) is significantly higher than that of the random integration monoclonal cell line (Random-Clone).

[0176] The results confirm that the site-specific integration system provided by this invention can significantly improve the integration efficiency and expression level of exogenous genes, and is superior to traditional random integration methods.

[0177] The above description of specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications based on the present invention, and as long as they do not depart from the spirit of the present invention, they should all fall within the scope of the appended claims.

Claims

1. A cellular genomic locus for targeted integration of exogenous nucleic acid molecules, wherein the locus is located in the nucleic acid sequence of the corresponding Chinese hamster ovary CHO-K1 cell genome NC_048595.1: 454997734-455024715.

2. The cell genomic locus according to claim 1, characterized in that, The cell genomic site is located in the nucleic acid sequence corresponding to the nucleotide sequence shown in SEQ ID NO: 1; Preferably, the cellular genomic site is located in the nucleic acid sequence corresponding to the nucleotide sequence shown in SEQ ID NO: 2; More preferably, the cell genomic site is located in the nucleic acid sequence corresponding to the nucleotide sequence shown in SEQ ID NO: 3; More preferably, the cell genomic site is located in the nucleic acid sequence corresponding to the nucleotide sequence shown in SEQ ID NO:

4.

3. The application of the cell genomic site described in claim 1 or 2 in the targeted integration of exogenous nucleic acid molecules.

4. The application according to claim 3, characterized in that, The targeted integration is achieved through homologous targeted repair (HDR); Preferably, the exogenous nucleic acid molecule is a DNA molecule, such as a single-stranded DNA molecule or a double-stranded DNA molecule, used to express one or more exogenous proteins; Preferably, the application further involves expressing one or more exogenous proteins in cells.

5. The application according to claim 3 or 4, characterized in that, The exogenous nucleic acid molecule comprises: a first recombinant recognition sequence and a second recombinant recognition sequence recognized by a recombinase, and one or more expression cassettes located between the first recombinant recognition sequence and the second recombinant recognition sequence, the expression cassettes comprising the exogenous nucleic acid sequence and a promoter driving its expression; Preferably, the exogenous nucleic acid sequence is a nucleic acid sequence or gene encoding a target protein (POI), a selection marker, and / or a reporter protein; More preferably, the exogenous nucleic acid molecule further comprises a 5' homologous arm and a 3' homologous arm, each comprising a nucleotide sequence homologous to the nucleic acid sequence surrounding the cellular genomic site as described in claim 1 or 2.

6. A nucleic acid molecule comprising: a first recombinant recognition sequence and a second recombinant recognition sequence recognized by a recombinase, and one or more expression cassettes located between the first recombinant recognition sequence and the second recombinant recognition sequence, the expression cassettes comprising a foreign nucleic acid sequence and a promoter driving its expression.

7. The nucleic acid molecule according to claim 6, characterized in that, The nucleic acid molecule is a DNA molecule, such as a single-stranded DNA molecule or a double-stranded DNA molecule; Preferably, the exogenous nucleic acid sequence is a nucleic acid sequence or gene encoding a target protein (POI), a selection marker, and / or a reporter protein; More preferably, the nucleic acid molecule further comprises a 5' homologous arm and a 3' homologous arm, each comprising a nucleotide sequence homologous to the sequence surrounding the cellular genomic site as described in claim 1 or 2.

8. A targeted integration cell, said targeted integration cell comprising the nucleic acid molecule of claim 6 or 7.

9. The method for preparing targeted integrated cells according to claim 8, wherein the preparation method comprises: The nucleic acid molecules are targeted and integrated into the host cell through homology-directed repair (HDR).

10. A method for producing protein, the method comprising: Culturing the targeted integration cells as described in claim 8, and collecting the proteins expressed by the nucleic acid molecules.

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