Library of genetically modified cell line having multiple genetic modifications and method for constructing same

Abstract

The present disclosure provides a library of a genetically modified cell line having multiple genetic modifications and a method for constructing the library.

Description

Library of genetically modified cell lines with multiple genetic modifications and method for constructing same

[0001] The present disclosure relates to libraries of genetically modified cell lines carrying multiple genetic modifications and methods for constructing the same.

[0002] Cell line libraries have been constructed, but in many cases, the cell lines contain only one genetic modification (Patent Documents 1 and 2).

[0003] WO2015 / 166272WO2014 / 085711

[0004] The present disclosure provides a library of genetically modified cell lines carrying multiple genetic modifications and methods for their construction.

[0005] According to the present disclosure, the following inventions are provided as examples: (1) A method for preparing a library of genetically modified cell lines, comprising: introducing different independent genetic modifications into an isolated cell line to thereby obtain a first library containing a plurality of first types of genetically modified cell lines; clustering the obtained first library into a plurality of different groups according to the cellular characteristics of each genetically modified cell line; selecting from each group a plurality of genetic modifications that produce the cellular characteristics, thereby listing the plurality of genetic modifications for each group; selecting two or more of the listed genetic modifications from one group, or selecting and combining one or more of the listed genetic modifications from each of a plurality of groups, to obtain a combination of genetic modifications containing two or more genetic modifications, and creating a cell line having the combination of genetic modifications, thereby obtaining a second library containing a second genetically modified cell line. (2) The method according to (1) above, wherein obtaining a combination of genetic modifications containing two or more genetic modifications comprises selecting and combining one or more of the listed genetic modifications from each of a plurality of groups. (3) The method according to (1) or (2) above, wherein the second library comprises at least 30 or more types of second genetically modified cell lines. (4) The method according to any one of (1) to (3) above, wherein the plurality of different groups comprises three or more, or four or more groups. (5) The method according to any one of (1) to (4) above, wherein the genetic modification comprises a genetic modification that has the potential to affect one or more abilities selected from the group consisting of metabolic ability, substance production ability, cell proliferation ability, cell differentiation ability, and the ability to maintain undifferentiated cell status. (6) The method according to any one of (1) to (5) above, wherein the cellular characteristics comprise the transcriptome. (7) The method according to any one of (1) to (6) above, wherein the genetic modification comprises one or more selected from the group consisting of additional gene introduction, gene mutation introduction, gene disruption, gene deletion, gene overexpression, and gene expression suppression. (8) The method according to any one of (1) to (7) above, wherein the genetic modification includes a genetic modification that may affect the substance production ability of the cell, the biochemical characteristics include transcriptome analysis or proteome analysis, and the genetic modification includes gene disruption or overexpression.(9) The method according to any one of (1) to (8) above, wherein the cells are selected from the group consisting of bacteria, fungi, algae, animal cells, and plant cells. (10) The method according to any one of (1) to (9) above, further comprising measuring one or more cellular characteristics for each of the second genetically modified cell lines contained in the obtained second library, and obtaining information correlating each of the second genetically modified cell lines with the one or more cellular characteristics. (11) The method according to (10), further comprising one or more modification cycles (where n is 2 in the first modification cycle, and n is incremented by 1 for each modification cycle) including further clustering the nth genetically modified cell line into multiple clusters based on the acquired information, combining all of the genetic modifications of two or more of the nth genetically modified cell lines contained in the same cluster or two or more different clusters, or combining all of the genetic modifications of the nth genetically modified cell line with all of the genetic modifications of the mth genetically modified cell line (where m is a natural number smaller than n), and further modifying the genetically modified cell line to contain the resulting combination of genetic modifications, thereby obtaining a third library containing the nth genetically modified cell line. (12) The method according to any one of (1) to (11), further comprising one or more modification cycles (where n is 2 in the first modification cycle, and n is incremented by 1 for each modification cycle).

[0006] An example of the process for producing a second library from a first library is shown. An example of the process for producing an (n+1)th library from an nth library is shown (n is a natural number equal to or greater than 2). A specific flow in an embodiment is shown. The flow can be linked to the flow in FIG. 4. A specific flow in an embodiment is shown. The flow can be linked to the flow in FIG. 5. A specific flow in an embodiment is shown.

[0007] <Definition of Terms> As used herein, "genetic modification" refers to a natural or non-natural modification of a gene. The modification is a change in the sequence of a gene. The modification of the sequence of a gene may include one or more selected from the group consisting of insertion, addition, deletion, and substitution of the entire or partial sequence of the gene. The gene may be present in the genome of a cell or in genetic material outside the genome. As used herein, a cell line into which a genetic modification has been introduced is referred to as a genetically modified cell line. In other words, a genetically modified cell line is a cell line that has a genetic modification. Genetic modification can be performed by conventional methods. For example, by using genome editing technology (CRISPR / Cas system, TALEN, zinc finger nuclease, etc.), it is possible to selectively modify desired locations in the genome. As the CRISPR / Cas system, the CRISPR / Cas9 system or the CRISPR / Cas3 system (see, for example, WO2018 / 225858A) can be used.

[0008] As used herein, a "cell" is the fundamental structural and functional unit of all living organisms, possessing a membrane-enclosed cytoplasm and a genome. Cells can be prokaryotic (e.g., bacterial) or eukaryotic (e.g., fungi, animals, and plants) cells. A "cell line" is a single-cell cloned cell from an organism, or a modified cell thereof, that typically maintains certain properties and can be stably grown over long periods of time. As used herein, a cell line also includes cells from a single-celled organism that have been single-cell cloned. Single-cell cloning refers to the isolation of a single cell from an original cell population and the creation of a genetically identical monoclonal cell population from that cell. Single-cell cloning can be achieved, for example, by limiting dilution of a cell population. For example, single-cell cloning can be performed by diluting a cell population to one cell or less per well (e.g., an average of 0.5 cells), and dispensing the resulting cell suspension into wells.

[0009] As used herein, a "library" refers to a collection of a variety of homogeneous but distinct substances, primarily prepared for screening. A library typically includes different containers (e.g., tubes, microtubes, and wells of a multi-well plate) each containing a variety of constituent substances. Tubes or microtubes can contain substances in volumes of, for example, 10 mL, 5 mL, 1.5 mL, 0.5 mL, 0.2 mL, or 0.1 mL. A microtube may be a combination of multiple microtubes. A multi-well plate includes, for example, a substrate and multiple wells (e.g., 96 wells or 384 wells) formed on the substrate. The multiple substances in a library are typically not mixed and are contained separately. A library of genetically modified cell lines includes cell lines with a variety of different genetic modifications. In a library of genetically modified cell lines, each genetically modified cell line is essentially physically isolated from all other genetically modified cell lines. A library of genetically modified cell lines typically includes more than several dozen genetically modified cell lines.

[0010] As used herein, "cellular characteristics" refer to characteristics possessed by cells, and include, for example, physical characteristics (e.g., size, shape, membrane hardness, membrane elastic modulus, brightness under an optical microscope, etc.), biochemical characteristics (e.g., protein expression, post-translational modification, RNA expression, activation of signal transduction pathways, etc.), and physiological characteristics (e.g., proliferation ability, division ability, metabolic ability, substance production ability, differentiation ability, undifferentiation ability (ability to maintain undifferentiation), adhesion ability, motility, etc.).

[0011] <Method for Preparing a Library> The present disclosure provides a library of genetically modified cell lines and a method for preparing the same. Genetically modified cell lines have been utilized for the production of useful substances. The genomes of various organisms have already been elucidated, making it relatively easy to disrupt or insert predicted genes. However, there are gene regions whose functions are unknown, and functional analysis of these gene regions remains difficult. Furthermore, because the number of types of genetically modified cell lines with multiple genetic modifications increases logarithmically with the number of modifications, it is often impractical to prepare all combinations.

[0012] When preparing a library, the direction of genetic modification can be changed by clarifying the intended use of the library. However, the intended use of the library does not necessarily have to be clear. Furthermore, specific genetic modifications may be selected based on some indicator, but they may also be random (e.g., mutations using chemicals, radiation, and CRISPR libraries). However, preferably, the mutation site is easily identified (specifically, site-specific mutagenesis). If the mutation site cannot be easily identified, a step of identifying the mutation site is carried out. The method will be performed in vitro.

[0013] A commonly used site-specific mutagenesis method is a mutation introduction method using homologous recombination. Homologous recombination involves an upstream homology arm (homologous) capable of recombining with the upstream of the mutation introduction site and a downstream homology arm (homologous) capable of recombining with the downstream of the mutation introduction site. By including an arbitrary sequence (the sequence length is 0, i.e., the sequence may be absent) between the upstream and downstream homology arms, an arbitrary sequence is inserted between the upstream and downstream of the mutation introduction site. If no sequence exists between the upstream and downstream homology arms, the region between the upstream and downstream of the mutation introduction site will be deleted. This homologous recombination is promoted at the DNA cleavage site. Therefore, it is beneficial to cleave the site that induces homologous recombination in a sequence-specific manner.

[0014] In eukaryotic cells, sequence-specific DNA cleavage triggers the repair mechanism for that DNA. Because the DNA repair mechanism is not precise, a certain percentage of genes are disrupted, resulting in gene-disrupted strains. The promotion of homologous recombination at the DNA cleavage site is also due to the DNA repair mechanism, but in the case of homologous recombination, a given sequence is precisely inserted between the upstream and downstream of the mutation introduction site (or precisely deleted if no given sequence is present).

[0015] Sequence-specific cleavage of DNA is possible using genome editing techniques (e.g., CRISPR / Cas systems, TALEN, zinc finger nucleases, etc.) and restriction enzymes (e.g., restriction enzymes that recognize and cleave 16 bases or more in length (e.g., 17 bases, 18 bases, etc.)). These cleavage techniques can be performed appropriately by those skilled in the art.

[0016] Recently, systems for gene modification using the CRISPR / Cas system and transposons have been reported (see Strecker, J., Science, Vol. 365, Issue 6448, pp. 48-53, 2019; and Klompe, SE, Nature, 571, 219-225, 2019, the entire contents of which are incorporated herein by reference). These systems are based on the discovery that transposons such as Tn7-like transposons (e.g., derived from cyanobacteria or Vibrio cholerae) contain CRISPR-associated genes (e.g., V-k type or IF type) in addition to transposon-associated genes. In these systems, Cas does not need to have DNA cleavage activity, but the sequence of the spacer region of the crRNA determines the insertion site of the transposon. In these systems, specific insertion of the transposon can be achieved by designing the spacer region of the crRNA, and these systems have already been utilized for gene transfer in bacteria. Therefore, in the present disclosure, a transposon (e.g., a Tn7-like transposon) having a transposon-associated gene and a CRISPR-associated gene may be used to introduce genetic modification. In this system, the inserting DNA may be linear or circular DNA (e.g., a plasmid) having transposase recognition sequences at both ends of the inserted sequence.

[0017] In the present disclosure, the method includes providing a collection of isolated cell lines having one or more genetic modifications. This cell line is referred to herein as a first genetically modified cell line, and this collection is referred to as a first library (see step S-1 in FIG. 1). The genetic modifications may typically be as defined above. As described above, specific genetic modifications may be selected based on some criteria, but may also be random. Preferably, the cell line is typically derived from a cloned cell line (i.e., a cell line having a single genetic background (isogenic genetic background)).

[0018] The first library may contain, but is not limited to, for example, 50 to 10,000 types (eg, 100 to 1,000) of first genetically modified cell lines.

[0019] In one aspect, the method may include measuring the cellular characteristics of each of the first genetically modified cell lines contained in the first library (see Figure 1, step S-2). The cellular characteristics may be any measurable property of a cell, and may be, for example, one or more selected from the group consisting of physical properties, biochemical properties, and physiological properties. Physical properties include, but are not limited to, cell size, shape (including three-dimensional shape such as shape and thickness), cell membrane stiffness, cell membrane elasticity, cell membrane permeability, brightness under an optical microscope, and the shape of aggregates formed with other cells (including the shape of clumps and layered structures). Biochemical properties include, but are not limited to, protein expression (proteome), post-translational modifications, RNA expression (transcriptome), metabolites (metabolome and fluxome), DNA modifications and histone modifications (epigenome), lipids (lipidome), glycans (glycome), intermolecular interactions (interactome), and activation of signaling pathways. Physiological properties include, but are not limited to, phenomes such as cell proliferation ability, division ability, metabolic ability, substance production ability, differentiation ability, undifferentiated ability (ability to maintain undifferentiated state), adhesion ability, motility ability, substrate reactivity (substrate decomposition ability, etc.), drug responsiveness (drug resistance, drug sensitivity, etc.), etc. Cell properties can be characterized by an increase or decrease in a specific indicator of any of these.

[0020] In one embodiment, the cell line may be a microorganism used in substance production (e.g., prokaryotes, fungi, and algae). Examples of prokaryotes include Escherichia coli, Bacillus subtilis (e.g., Bacillus sp.), lactic acid bacteria (e.g., Lactobacillus sp.), actinomycetes (e.g., Corynebacterium sp., Streptomyces sp., Bifidobacterium sp.), and hydrogen-oxidizing bacteria (e.g., Alcaligenes eutrophus, Hydrogenophaga pseudoflava, Paracoccus denitrificans, etc.). Fungi include Aspergillus oryzae, yeast, and Trichoderma reesei. In this embodiment, genetic modification may preferably include modification of genes related to metabolic pathways (e.g., enzymes in metabolic pathways and proteins that control these enzymes). By enhancing the expression of a specific gene and / or suppressing the expression of another specific gene, the production of a specific metabolic product may be increased.

[0021] In some aspects, the cell line is selected from yeast (e.g., fission yeast or budding yeast, e.g., Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Saccharomyces fragilis, Saccharomyces rouxii, and the like), Candida utilis, Candida tropicalis, and the like). In some embodiments, the cell line may be an organism selected from the group consisting of Candida species such as Candida tropicalis, Pichia, Kluyveromyces, Yarrowia, Hansenula, and Endomyces. In some aspects, the cell line may be an actinomycete (e.g., Streptomyces genus, e.g., Streptomyces lividans, Streptomyces griseus, Streptomyces aurefaciens, Streptomyces rimosus, Nocardia genus, e.g., Nocardia asteroides, Nocardia spp. ... The cell strain is a bacterium of the genus Actinomyces, such as Actinomyces spiralis. In one embodiment, the cell strain is a Bacillus subtilis (e.g., Bacillus subtilis). In one embodiment, the cell strain is a hydrogen bacterium. Hydrogen bacteria are bacteria that have the ability to oxidize hydrogen and perform carbon dioxide assimilation using the energy generated by the oxidation reaction (also referred to as "hydrogen-oxidizing bacteria"). Hydrogen bacteria (e.g., aerobic hydrogen bacteria, Knallgas bacteria) can use hydrogen as an electron donor and oxygen as an electron acceptor, and have the ability to reductively assimilate carbon dioxide to synthesize organic matter. Hydrogen bacteria are not particularly limited, but examples include bacteria of the genus Caminibacter, bacteria of the genus Aquifex, bacteria of the genus Ralstonia, and bacteria of the genus Paracoccus.Examples of hydrogen bacteria include bacteria of the genus Alcaligenes, Pseudomonas, Bacillus, and Hydrogenobadcter. Hydrogen bacteria can grow well in the presence of sufficient nutrients, hydrogen, oxygen, and carbon dioxide. Because hydrogen bacteria produce substances such as proteins from carbon dioxide, they are effective in reducing carbon dioxide emissions and can produce organic substances such as proteins (e.g., animal proteins), food, plastics, chemicals, and biofuels, making them highly anticipated for use. Hydrogen bacteria preferably have a group 1h / 5 hydrogenase. In a preferred embodiment, the hydrogen bacteria may be Ralstonia eutropha.

[0022] In one aspect, the cell line may be a mammalian cell line. In one embodiment, the mammalian cultured cell line may be derived from any human tissue, such as the stomach, small intestine, large intestine, lung, pancreas, kidney, liver, thymus, spleen, prostate, ovary, uterus, bone marrow, skin, muscle, or peripheral blood. In one embodiment, the human-derived cells used in the present invention are non-blood cells. The human-derived cells used in the present invention may be derived from specific cell types in human tissues (e.g., epithelial cells, endothelial cells, epidermal cells, stromal cells, fibroblasts, adipose tissue, mammary cells, mesangial cells, pancreatic beta cells, neurons, glial cells, exocrine epithelial cells, endocrine cells, skeletal muscle cells, smooth muscle cells, cardiac myocytes, osteoblasts, embryonic cells, immune cells (B cells, T cells, NK cells, NKT cells, macrophages, neutrophils, basophils, eosinophils, dendritic cells), etc.). The human-derived cells used in the present invention may be normal cells or cancer cells. In one embodiment, the human-derived cells used in the present invention are normal cells (eg, primary cells, such as primary somatic cells).

[0023] In one embodiment, the human-derived cells used in the present invention may be human embryonic kidney 293 (HEK293) cells (J. Gen. Virol.; 1977; 36: 59-74), HCT116 cells, WI-38 cells, SC-01MFP cells, or MRC-5 cells, as well as cells derived from these cells. In one embodiment, the human-derived cells used in the present invention may be cells derived from HEK293 cells (FreeStyle TM 293-F cells, etc. In one embodiment, the mammalian cells used in the present disclosure may be, for example, Chinese hamster ovary cells (CHO cells).

[0024] In one aspect, the cell line may be a human pluripotent cell (e.g., an embryonic stem cell (ES cell), an induced pluripotent stem cell (iPS cell), or an epiblast stem cell). Pluripotent stem cells include naive and primed pluripotent stem cells. In one aspect, the cell line is not a human germline cell (spermatogonia, sperm, oocyte, or egg), a human fertilized egg, or an early human embryo.

[0025] In one aspect, the method further comprises clustering the resulting first library into multiple different groups according to the cellular characteristics of each genetically modified cell line (see step S-3 in Figure 1). Clustering typically involves classifying cells with similar cellular characteristics into the same group and cells with different cellular characteristics into different groups. The number of cell lines included in the same group varies depending on the degree of similarity, but the degree of similarity can be determined so that the entire library can be clustered into approximately 5 to 20 groups, for example. Cells that do not belong to any group may be grouped together into a miscellaneous group.

[0026] Clustering methods include, but are not limited to, hierarchical clustering (e.g., Ward's method, centroid method, group average method, shortest distance method, median method, etc.), K-means method, principal component analysis (PCA), t-SNE (t-SNE), Uniform Manifold Approximation and Projection (UMAP), and graph-based clustering (e.g., Louvain / Leiden Community Detection), and can be used as appropriate. Clustering methods also include those using machine learning (including deep learning-based clustering). Deep learning-based clustering includes autoencoder clustering, deep embedded clustering (DEC), and attention-based clustering, as well as modified versions of these.

[0027] In the method of the present disclosure, after clustering, one or more strains to be included in each cluster are selected. The selected strains have specific genetic modifications. The selected genetic modifications can be combined with genetic modifications within the cluster or with genetic modifications from other clusters. For example, a genetic modification selected from the nth cluster can be combined with another genetic modification selected from the nth cluster. In a preferred embodiment, a genetic modification selected from the nth cluster can be combined with another genetic modification selected from the mth cluster. n and m are different natural numbers equal to or less than the number of clusters. In this embodiment, another genetic modification selected from a further cluster may be further combined. That is, genetic modifications possessed by strains included in two or more, or three or more clusters may be combined (see step S-4 in Figure 1).

[0028] The method of the present disclosure may further include preparing a second genetically modified cell line having a combination of the above genetic modifications, thereby preparing a second library containing the second genetically modified cell line (see step S-5 in Figure 1).

[0029] The second library may contain, but is not limited to, for example, 100 to 100,000 types (e.g., 100 to 10,000) of second genetically modified cell lines. The types of second genetically modified cell lines contained in the second library (i.e., the size of the second library) are preferably larger than the types of first genetically modified cell lines contained in the first library (i.e., the size of the first library), and are not limited to, for example, 10 times or more, 15 times or more, 20 times or more, 25 times or more, 30 times or more, 40 times or more, 50 times or more, 60 times or more, 70 times or more, 80 times or more, 90 times or more, or 100 times or more (e.g., 10 times to 500 times, e.g., 20 to 100 times). In a preferred embodiment, the combination of genetic modifications enhances some or all of the cellular characteristics, and the size of the second library can be efficiently expanded to include many cell lines with enhanced cellular characteristics.

[0030] In the method of the present disclosure, a second library can be prepared as described above. The method of the present disclosure may also include measuring additional cellular characteristics of each of the second genetically modified cell lines contained in the second library. By measuring the cellular characteristics resulting from multiple genetic modifications in each cell line, a large data set correlating the multiple genetic modifications with the resulting cellular characteristics can be obtained. This data can be used to derive additional combinations of genetic modifications or to select cell lines suitable for the purpose from the second library.

[0031] The combination of genetic modifications can be further optimized by further clustering, selecting strains from each cluster, identifying the genetic modifications carried by the strains, and creating additional strains carrying all of the genetic modifications carried by multiple strains. That is, the method of the present disclosure may further include one or more modification cycles (where n is 2 in the first modification cycle, and n is incremented by 1 each time a modification cycle is performed) that include further clustering the nth genetically modified cell line into multiple clusters based on the acquired information, combining all of the genetic modifications carried by two or more of the nth genetically modified cell lines contained in the same cluster or two or more different clusters, and further modifying the genetically modified cell line to contain the resulting combination of genetic modifications, thereby obtaining a third library containing the n+1th genetically modified cell line (where n is 2 in the first modification cycle, and n is incremented by 1 each time a modification cycle is performed) (see FIG. 2). The step of combining all of the genetic modifications carried by two or more nth genetically modified cell lines contained in the same cluster or two or more different clusters may be replaced by combining all of the genetic modifications carried by the nth genetically modified cell line with all of the genetic modifications carried by the mth genetically modified cell line (where m is a natural number smaller than n). By increasing the number of modification cycles, it may be possible to arrive at a combination of genetic modifications optimized for a specific purpose. The nth genetically modified cell line may contain, for example, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more different genetic modifications (where n is a natural number greater than or equal to two). Cell lines that fail to grow and / or die in any of the above steps may not be included in the library. In some aspects, the nth library includes all combinations of the listed genes, or a subset thereof (e.g., 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of all combinations).

[0032] <Library of the Present Disclosure> The present disclosure provides a library of genetically modified cell lines (e.g., an nth library) obtained by the above-described method. This library may be useful in various screening procedures.

[0033] <Application Example> A specific application example of the library preparation method described above will be described below. In this application example, an attempt is made to obtain a library containing genetically modified cell lines that enhance substance production. Omics data may already be obtained, and the function of each gene may be estimated. In such cases, for example, 300 types of genes (referred to as regulatory genes here) that are suggested to have the potential to affect substance production or are highly likely to do so based on the omics data (which may be publicly available) are selected (see Design_1 in Figure 3). Examples of omics data include, but are not limited to, gene annotation data, comprehensive transcription analysis data (e.g., RNA-Seq data), and protein-protein interaction (PPI) data.

[0034] Next, genetic modifications are introduced into the target substance-producing strain (parent strain) (see Build_1 in Figure 3). In this process, cell lines can be prepared in which each of the 300 selected genes is deleted and / or overexpressed. A total of 600 cell lines with each of the genetic modifications are obtained, and each is subjected to analysis of cellular characteristics (transcriptome analysis is exemplified here). Based on transcriptome similarity, the cells are clustered into, for example, 5 to 10 groups (8 in the figure) (see Learn_1 in Figure 3).

[0035] Next, a multiply genetically modified cell line is created (see DBTL2 in Figure 3). While selecting multiple genetic modifications from the same group is not excluded, in Figure 3, multiple genetic modifications are selected from multiple groups to obtain a group of cell lines with all of the mutations (see Design & Build_2 in Figure 3). For example, one genetic modification can be selected from each of two groups and combined to obtain a cell line with these two genetic modifications, or one genetic modification can be selected from each of three groups and combined to obtain a cell line with these three genetic modifications. This allows for the creation of a second library. The number of genetic modifications to be combined is not limited to two or three. In the second library, the number of genetic modifications in each cell line does not need to be the same. That is, the same library may contain genetically modified cell lines with different numbers of genetic modifications.

[0036] Each cell line (second genetically modified cell line) contained in the resulting second library can be subjected to cell characteristic analysis, in which the amount of target substance produced is measured (see Figure 3, test_2). A data set linking the genetic modifications and the amount of target substance produced can be obtained for each cell line (see Figure 3, learn_2). At this stage, superior strains can be used to produce the target substance. Figure 3 also proposes a further genetic modification process.

[0037] Each of the second genetically modified cell lines can be subjected to cell characterization analysis. This involves measuring environmental factors, such as culture conditions (media composition and other conditions), growth rate, transcriptome analysis, metabolome analysis, and flux analysis (see Figure 4). A dataset can be generated by linking the genetic modifications to the relationship between these measurements and the amount of target substance produced.

[0038] Clustering is performed based on the dataset obtained in Figure 4. Figure 5 proposes clustering using principal component analysis and cluster analysis, or machine learning. This allows for the derivation of a combination of genetic modifications that maximizes the production of the target substance. Genetically modified cell lines with the derived combinations can be obtained to construct a further library (third library). The library can also be screened to identify superior strains for use in the production of the target substance. Figure 5 also proposes repeating the Learn_3 step. In this way, the library can ultimately be screened to identify superior strains for use in the production of the target substance.

[0039] In one aspect, the library may be provided together with cell characteristic information associated with each genetically modified cell line contained therein, or a recording medium (non-volatile or volatile) containing such information.

[0040] Practical Application Example 1 In one embodiment, the cell line is Escherichia coli. In Escherichia coli, the number of protein-encoding genes is approximately 4,000. Techniques for synthesizing various proteins or metabolites using Escherichia coli are known, and the method of the present disclosure is utilized for substance production using Escherichia coli.

[0041] Practical Application Example 2 In one embodiment, the cell strain is a hydrogen-oxidizing bacterium. In hydrogen-oxidizing bacteria, the number of protein-encoding genes is approximately 6,000. Techniques for synthesizing various proteins or metabolites using hydrogen-oxidizing bacteria are known, and the method of the present disclosure can be utilized for substance production using hydrogen-oxidizing bacteria. For example, the method can be utilized for the production of polyhydroxybutyrate, e.g., poly-[(R)-hydroxybutyrate].

[0042] Practical Application Example 3 In one aspect, the cell line is a mammalian cultured cell line. In one aspect, the method of the present disclosure can be utilized to produce proteins (e.g., secreted factors, cytokines, hormones, enzymes, antibodies, etc.) using this cell line. The produced proteins can be administered to humans. For diploid cells, it may be desirable to simultaneously modify two alleles on the genome for genetic modification. Such modifications can be efficiently achieved, for example, using the technology (UKiS) disclosed in WO 2021 / 206054.

[0043] Practical Application Example 4 In one aspect, the cells are immune cells. Immune cells are used in immunotherapy and can be made hypoimmunogenic (universalized) so that they can be administered to allogeneic subjects. For example, allogeneic therapeutic techniques using chimeric antigen receptor (CAR)-expressing immune cells (e.g., T cells, NK cells, NKT cells, macrophages, etc.) usually require hypoimmunogenic cells for CAR introduction. By gene disruption or gene introduction, the method of the present disclosure can be useful for constructing immune cell platforms for various therapies.

[0044] Practical Application Example 5 In one embodiment, the cells are pancreatic beta cells. Cells that secrete insulin in response to glucose are sought after for the treatment of diabetes through regenerative medicine. Administration to allogeneic subjects requires hypoimmunogenic cells. Through gene disruption or gene transfer, the disclosed method may be useful for constructing a beta cell platform for various therapeutic applications.

[0045] The above practical applications can be broadly expanded beyond the specific cells exemplified. The method of the present disclosure is particularly useful for improving substance production processes by modifying microorganisms and establishing cell therapy (including immunotherapy) techniques by modifying cells.

Claims

1. A method for preparing a library of genetically modified cell lines, comprising: introducing different independent genetic modifications into an isolated cell line, thereby obtaining a first library containing a plurality of types of first genetically modified cell lines; clustering the obtained first library into a plurality of different groups according to the cellular characteristics of each genetically modified cell line; selecting from each group a plurality of genetic modifications that produce the cellular characteristics, respectively, to list a plurality of genetic modifications for each group; selecting two or more of the listed genetic modifications from one group, or selecting and combining one or more of the listed genetic modifications from each of a plurality of groups, to obtain a combination of genetic modifications containing two or more genetic modifications, and creating a cell line having the combination of genetic modifications, thereby obtaining a second library containing second genetically modified cell lines.

2. The method of claim 1, wherein obtaining a combination of genetic modifications comprising two or more genetic modifications comprises selecting and combining one or more of the listed genetic modifications from each of a plurality of groups.

3. The method of claim 1 or 2, wherein the second library comprises at least 30 or more second genetically modified cell lines.

4. The method of any one of claims 1 to 3, wherein the plurality of different groups comprises three or more, or four or more groups.

5. The method according to any one of claims 1 to 4, wherein the genetic modification comprises genetic modification that has the potential to affect one or more abilities selected from the group consisting of metabolic ability, substance production ability, cell proliferation ability, cell differentiation ability, and ability to maintain undifferentiated cell status in cells.

6. The method of any one of claims 1 to 5, wherein the cellular characteristics include the transcriptome.

7. The method according to any one of claims 1 to 6, wherein the genetic modification comprises one or more selected from the group consisting of additional gene introduction, gene mutation introduction, gene disruption, gene deletion, gene overexpression, and gene expression suppression.

8. The method according to any one of claims 1 to 7, wherein the genetic modification comprises a genetic modification that has the potential to affect the substance production ability of the cell, the biochemical characteristics comprise transcriptome analysis or proteome analysis, and the genetic modification comprises gene disruption or overexpression.

9. The method of any one of claims 1 to 8, wherein the cell is selected from the group consisting of a bacterium, a fungus, an alga, an animal cell, and a plant cell.

10. The method of any one of claims 1 to 9, further comprising measuring one or more cellular characteristics for each second genetically modified cell line contained in the obtained second library, and obtaining information relating each second genetically modified cell line to the one or more cellular characteristics.

11. The method of claim 10, further comprising one or more modification cycles, including further clustering the nth genetically modified cell line into multiple clusters based on the acquired information, combining all of the genetic modifications carried by two or more of the nth genetically modified cell lines contained in the same cluster or two or more different clusters, or combining all of the genetic modifications carried by the nth genetically modified cell line with all of the genetic modifications carried by the mth genetically modified cell line (where m is a natural number smaller than n), and further modifying the genetically modified cell line to contain the resulting combination of genetic modifications, thereby obtaining a third library containing the nth+1th genetically modified cell line (where n is 2 in the first modification cycle, and n is incremented by 1 each time a modification cycle is performed).

12. A library of genetically modified cell lines obtained by the method of any one of claims 1 to 11.

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