Genetically manipulated cells

Abstract

The present invention relates to cultured animal cells having a genetic modification in the NF2 gene or having a modification to Merlin protein activity wherein the animal is an animal species suitable for human or animal consumption. Also provided are methods of producing cultured meat or cultured meat products 5 comprising culturing animal cells having such mutations.

Description

Technical Field

[0001] The present invention relates to cultured meat cells for genetic manipulation. More specifically, the present invention relates to cultured meat cells that have been genetically manipulated to reduce the cell doubling time. Background Art

[0002] In the next 50 years, the world's population will increase to nearly 10 billion people. Therefore, by 2050, nearly 2 billion more people will need to be fed. By 2050, population growth will lead to an increase in global meat demand of about 73%. The agricultural industry will have to expand, possibly doubling in size, to meet this demand. Currently, 39% of the habitable land on Earth is used to produce feed for livestock used in the meat industry. It takes three years to raise a cow for slaughter, and 6 - 12 months for pigs and poultry. Therefore, large areas of arable land are needed to raise these animals. Currently, 80 billion animals are slaughtered annually for meat, and 1.2 billion animals are slaughtered in the UK alone.

[0003] Cultured meat has the potential to solve major global problems related to livestock farming, the environmental impact of meat production, and animal welfare, food security, and human health. Cultured meat is meat produced by in vitro cell culture of animal cells. This is a form of cellular agriculture that explores this agricultural method in the context of increasing consumer demand for protein. Cellular agriculture involves the production of animal - derived foods through cell culture.

[0004] Cultured meat is produced using tissue engineering techniques traditionally used in regenerative medicine and requires cell lines, usually stem cells. Stem cells are undifferentiated cells that have the potential to become many or all of the desired specialized cell types. Although pluripotent stem cells are generally considered the ideal starting cells, the most prominent example in this subclass of stem cells is embryonic stem cells, whose use in research is controversial due to ethical issues. Therefore, induced pluripotent stem cells (iPSCs) have been developed. iPSCs are pluripotent blood cells and pluripotent skin cells that are artificially reverted to a pluripotent state, enabling them to differentiate into a wider range of cells. Alternatives to iPSCs involve the use of multipotent adult stem cells that give rise to the muscle cell lineage or unipotent progenitor cells that can differentiate into muscle cells. The favorable properties of stem cells make them suitable for cultured meat production, and these favorable properties include immortality, enhanced proliferative capacity, non - dependence on adherence, serum independence, and ease of differentiation into tissues.

[0005] Stem cells for generating cell lines can be collected from primary sources, i.e., by performing a biopsy on an animal under local anesthesia, or established from secondary sources such as cryopreserved cultures. However, somatic cells isolated from tissues / organs (such as muscle, fat, and fibroblasts) commonly used for consumption in agriculturally relevant species (such as pigs, cows, chickens) have a limited lifespan when grown in vitro. Although primary cell lines (myoblasts, myofibroblasts, fibroblasts, adipose-derived stem cells, and epithelial cells) can be isolated from pigs, the ability to proliferate these cell lines with a high doubling time and long-term is not feasible.

[0006] Merlin (moesin-ezrin-radixin-like protein, also known as schwannomin) is a tumor suppressor protein encoded by the neurofibromatosis type 2 gene NF2. The NF2 gene and the Merlin protein play a central role in many important developmental signaling pathways. For example, NF2 / Merlin is a known activator of the Hippo pathway, which limits organ size (Hamaratoglu et al., 2006 (doi:10.1038 / ncb1339)), and plays a role in anoikis (Zhao et al., 2012; doi:10.1101 / gad.1733435.111). NF2 / Merlin has also been shown to regulate Ras activity in mouse cells (Cui et al., 2019 (doi:10.1038 / s41388-019-0883-6)). In addition, it is involved in WNT / beta-catenin signaling, receptor tyrosine kinase signaling, and the NOTCH signaling pathway, etc. (Mota & Shevde 2020 (doi.org / 10.1186 / s12964-020-00544-7). NF2 also plays a driving role in allowing cancer cells to escape from the primary tumor niche by downregulating cell-cell adhesion, thus promoting metastasis (Lallemand et al., 2003; doi:10.101 / gad.1054603). NF2 knockout models in various mouse and human cell lines show increased cell proliferation and loss of contact inhibition (Wahiduzzaman et al., 2018 (doi:10.1111 / cas.13871); Bosco et al., 2010 (doi:10.1038 / onc.2010.20); and Fomicheva & Macara, 2020 (doi:10.7554 / eLife.636032).

[0007] There is a need to reduce the cell doubling time (also in the context of suspension culture) to improve the economic feasibility of cultured meat. The present invention meets this need by providing primary animal cells that contain an endogenous genetic modification and do not require the manipulation of expressing an exogenous nucleic acid construct. To this end, the relative effects of the loss of Hippo pathway members and regulators on the proliferation of different cell types and genetic backgrounds in an anchorage-dependent and independent manner were compared. Summary of the Invention

[0008] In a first aspect of the present invention, there are provided cultured animal cells in which the expression of the NF2 gene is modified or the activity of the Merlin protein is modified, and wherein the animal is an animal species suitable for human or animal consumption. For the first time herein, it has been described that the functional knockout of endogenous NF2 reduces the doubling time of porcine and bovine myoblasts and adipose-derived stem cells. Additionally, studies have shown that the alteration of NF2 supports the suspension adaptation process of adherent porcine and bovine cell cultures, which is crucial for the production of agriculturally relevant cell types for consumption. Thus, it has been advantageously demonstrated that the inactivation of the endogenous NF2 gene is sufficient to accelerate the proliferation of porcine and bovine cell cultures and support the suspension adaptation process. Through the above advantages of increased proliferation (i.e., reduced doubling time) and support for the suspension adaptation process, the commercial feasibility of cultured meat is greatly improved.

[0009] In one embodiment, the modification reduces the doubling time of the cells by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.

[0010] In one embodiment, the animal is selected from pigs, cows, poultry, sheep, goats, equines, camels, fish, crustaceans or mollusks.

[0011] In one embodiment, the animal cells are somatic cells.

[0012] In one embodiment, the animal cells are selected from one of the following cell types: myoblasts, fibroblasts, myofibroblasts, adipose-derived stem cells, epithelial cells, mesenchymal stem cells, satellite cells, iPSCs or hepatocytes.

[0013] In one embodiment, in the animal cells, the expression of the NF2 gene or the activity of the Merlin protein is modified by any one or more of the following:

[0014] 1) Modification at the gene level by:

[0015] a. Knocking out or reducing the activity / transcription / translation level by editing the coding sequence, promoter, intron, regulatory region;

[0016] b. RNA-guided DNA methylation; or

[0017] c. Transcriptional activation or repression using CRISPRa or CRISPRi or similar target-specific methods;

[0018] d. Knockout or reduction of activity / transcription / translation levels by non-directed means, such as radiation or chemical mutagenesis;

[0019] 2) Modifications at the post-transcriptional level (post-transcriptional gene silencing) are carried out by the following methods:

[0020] a. RNAi or siRNA to reduce the translation of mRNA into protein; or

[0021] b. Site-specific nucleases to modify or cleave mRNA, such as CRISPR / Cas13a;

[0022] 3) Modifications at the post-translational level (protein disruption) are carried out by the following methods:

[0023] a. Containing activity-blocking / reducing molecules, where the activity-blocking / reducing molecules are small molecules, antibodies, etc.; or

[0024] b. Containing protein degradation components, where the protein degradation components are specialized proteases, exoproteases or endoproteases.

[0025] In one embodiment, the animal cell has a genetic modification in the NF2 gene.

[0026] In one embodiment, the genetic modification in the NF2 gene is a loss-of-function modification or results in reduced function.

[0027] In one embodiment, the loss-of-function modification includes gene knockout or loss of protein function.

[0028] In one embodiment, targeted genomic modification or random mutagenesis or modification by spontaneous mutation is used.

[0029] In one embodiment, the modification is located in the promoter region or coding region of one or more genes.

[0030] In one embodiment, targeted genomic modification is used, optionally using a targeted endonuclease to introduce the modification.

[0031] In one embodiment, the endonuclease is optionally selected from TALEN, ZFN or CRISPR, optionally CRISPR / Cas9.

[0032] In one embodiment, the cultured animal cells further comprise at least one additional genetic modification to manipulate genomic surveillance, cell cycle control, and / or cell death control pathways. In one embodiment, the at least one additional genetic modification is present in one or more of the following genes: RB1, TP53, and / or the RAS gene. In one embodiment, the additional genetic modification can be a knockout or reduced activity / transcription / translation level by editing the coding sequence, promoter, intron, regulatory region (e.g., in the RB1 or TP53 gene), or in another embodiment, the genetic mutation can be an insertion or increased activity / transcription / translation level by editing the coding sequence, promoter, intron, regulatory region (e.g., in the RAS gene).

[0033] In one embodiment, the animal cell has a genetic modification in RB1.

[0034] In one embodiment, the animal cell has a genetic modification in TP53.

[0035] In one embodiment, the animal cell has a genetic modification in the RAS gene.

[0036] In one embodiment, the animal cell has genetic modifications in RB1 and TP53.

[0037] In one embodiment, the animal cell has genetic modifications in RB1 and the RAS gene.

[0038] In one embodiment, the animal cell has genetic modifications in TP53 and the RAS gene.

[0039] In one embodiment, the animal cell has genetic modifications in RB1, TP53, and the RAS gene.

[0040] In one embodiment, the RAS gene is HRAS, NRAS, or KRAS.

[0041] In one embodiment, the RAS gene is HRAS.

[0042] According to a second aspect of the present invention, there is provided a method for producing cultivated meat or cultured meat products, which comprises culturing the animal cells described herein.

[0043] According to a third aspect of the present invention, there is provided a method for producing the modified animal cells as described herein.

[0044] In one embodiment, the animal is selected from pigs, cows, poultry, sheep, goats, equids, camelids, fish, crustaceans, or mollusks.

[0045] In one embodiment, the animal cells are somatic cells.

[0046] In one embodiment, the animal cells are selected from one of the following cell types: myoblasts, fibroblasts, myofibroblasts, adipose-derived stem cells, epithelial cells, mesenchymal stem cells, satellite cells, iPSCs, or hepatocytes.

[0047] In one embodiment, the modification is introduced using targeted genome editing.

[0048] According to a fourth aspect of the present invention, there is provided a cultured or cultivated animal tissue or a cultured or cultivated meat product comprising the modified cells as described herein.

[0049] In one embodiment, the culturing is suspension culture.

[0050] According to a fifth aspect of the present invention, there is provided the use of the modified animal cells as described herein for cell agriculture.

[0051] According to a sixth aspect of the present invention, there is provided a method for producing an immortalized animal cell line, which comprises the method as described herein, wherein the immortalized cell line comprises an NF2 gene modification.

[0052] In one embodiment, the modification is a loss-of-function modification or results in reduced function.

[0053] In one embodiment, the loss-of-function modification is a knockout of the NF2 gene.

[0054] According to a seventh aspect of the present invention, there is provided a guide RNA which targets the sequence of SEQ ID NO.5, or SEQ ID NO.8, or SEQ ID NO.9, or SEQ ID NO:10, or SEQ ID NO.28, or SEQ ID NO.29 or SEQ ID NO.30 alone or in combination.

[0055] According to an eighth aspect of the present invention, there is provided a kit comprising at least one guide RNA as described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] FIGURES AND TABLES

[0057] The present invention is further described in the following non-limiting figures.

[0058] Figure 1 is a graph showing the doubling time under suspension conditions. The doubling time was evaluated after transferring the control cell pool (transfected with Cas9 only, no sgRNA) and the NF2 knockout cell pool from adherent growth conditions to suspension growth conditions. Data points were from triplicate conical flasks and were measured over 4 growth passages.

[0059] Figure 2 shows the Figure 1 collective data of all 4 passages for doubling time assessment.

[0060] Figure 3 is a graph showing the NF2 gene editing efficiency. Assessment of NF2 knockout efficiency in a pool of porcine myoblast cells transfected with Cas9 protein and sgRNA targeting the NF2 gene. After 4 generations of suspension growth studies at two time points after transfection and in triplicate flasks, DNA was isolated from the cell pool, amplified by PCR of the NF2 gene, and then the PCR amplicons were Sanger sequenced, and the sequencing results were analyzed using the ICE analysis tool (ICE.synthego.com) to determine the knockout efficiency.

[0061] Figure 4 shows that functional knockout of the NF2 gene encoding the Merlin protein preserves the mesenchymal cell phenotype and enhances the adipogenic differentiation potential of immortalized myoblast cell lines. (a) Flow cytometry density plots of cell surface markers CD29 (mesenchymal), CD56 (myoblast), CD90 (mesenchymal), CD29 CD56+ / + (muscle stem cells), CD31 (endothelial cells), and CD45 (immune lineage), demonstrating loss of CD56 and CD90 expression in NF2(+ / +) cells after adaptation from adherent culture conditions to suspension, while CD56 and CD90 expression is retained after functional knockout of the NF2 gene. (b - g) Quantification of flow cytometry data for the corresponding surface markers in NF2(+ / +, purple bars) and (- / -, green bars) suspension cell lines. (h) Fluorescence micrographs of NF2(+ / +) and NF2(- / -) cell lines labeled with the lipid marker BODIPY (green) and nuclear DNA (DAPI, blue) in suspension show enhanced adipogenic potential in the NF2 knockout line, corresponding to the retained mesenchymal cell phenotype. Optical micrographs of NF2(- / -) cells also show unilocular nuclear localization indicating mature adipocytes (indicated by white arrows). (i) Quantification of lipid accumulation in NF2(+ / +) and (- / -) cell lines.

[0062] Figure 5 shows the NF2 knockout efficiency in various cell types. (a) Porcine primary myoblasts. (b) Porcine CRISPR - immortalized (P53 - / - / RB1 - / - / HRAS G12V / - ) myoblasts. (c) Porcine virus - immortalized myoblasts. (d) Porcine virus - immortalized ADSC (suspension). (e) Porcine CRISPR - immortalized (P53 - / - / RB1 - / - / HRASG12V / - ) ADSC. (f) Angus cattle variant CRISPR-immortalized (P53 - / - / RB1 - / - ) ADSC. (g) Angus cattle variant CRISPR-immortalized (P53 - / - / RB1 - / - / HRAS G12V / - ) ADSC. (h) Angus cattle variant CRISPR-immortalized (P53 - / - / RB1 - / - ) Myoblast. (i) Angus cattle variant CRISPR-immortalized (P53 - / - / RB1 - / - / HRAS G12V / - ) Myoblast. (j) Wagyu cattle variant CRISPR-immortalized (P53 - / - / RB1 - / - ) ADSC. (k) Wagyu cattle variant CRISPR-immortalized (P53 - / - / RB1 - / - / HRAS G12V / - ) Myoblast.

[0063] Figure 6 Shows the doubling time assessment of the NF2 knockout cell line compared to the control line under adherent growth conditions. (a) Doubling time of Angus cattle variant myoblasts (adherent). (b) Doubling time of Angus cattle variant myoblasts (adherent). (c) Doubling time of Angus cattle variant ADSCs (adherent). (d) Doubling time of Angus cattle variant ADSCs (adherent). (e) Doubling time of Wagyu cattle variant myoblasts (adherent), (f) Doubling time of Wagyu cattle variant myoblasts (adherent).

[0064] Figure 7 Shows the doubling time assessment of the NF2 knockout cell line compared to the control line under suspension growth conditions. (a) Doubling time of porcine myoblasts (suspension). (b) Doubling time of porcine myoblasts (suspension). (c) Doubling time of porcine ADSCs (suspension). (d) Doubling time of porcine ADSCs (suspension). (e) Doubling time of Angus cattle variant myoblasts (suspension). (f) Doubling time of Angus cattle variant myoblasts (suspension).

[0065] Figure 8 Compares the enrichment of cells with different gene mutations in the Hippo pathway in the growth competition assay.

[0066] Table 1 Sequences of nucleic acids. These sequences include the target sequences according to the present invention and illustrate the gene modifications. Detailed implementation mode

[0067] Detailed description

[0068] Aspects of the present invention will now be further described. In the following paragraphs, different aspects will be described. Each aspect so defined can be combined with any other one or more aspects, unless there is an express contrary indication.

[0069] Generally, the terms and techniques related to cell and tissue culture, pathology, oncology, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well-known and commonly used in the art. Unless otherwise noted, the methods and techniques of the present disclosure are generally performed according to conventional methods well-known in the art and described in various general and more specific references cited and discussed in this specification. For example, see Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012).

[0070] As is typically practiced in the art or as described herein, enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions. The terms related to cell biology and cell culture, analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein, as well as laboratory procedures and techniques, are well-known and commonly used in the art. Standard techniques are used for any cell culture, gene targeting, chemical synthesis, chemical analysis, and delivery.

[0071] Low cell doubling time and growth under suspension conditions are crucial for large-scale cultured meat processing. Somatic cells isolated from tissues / organs (such as muscle, fat, fibroblasts) commonly used for consumption from agricultural-related species (such as pigs, cows, chickens) have limited proliferative capacity when grown in vitro. In addition, cells isolated from animal tissues tend to grow only under adherent conditions, and transferring to suspension conditions results in very slow (if any) cell growth.

[0072] Modified cells and methods

[0073] In a first aspect, the present invention relates to cultured animal cells, wherein the expression of the NF2 gene is modified or the activity of the Merlin protein is modified, and wherein the animal is an animal species suitable for human or animal consumption.

[0074] As used herein, the terms "nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule", or "polynucleotide" are intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), naturally occurring, mutated, synthetic DNA or RNA molecules, and analogs of DNA or RNA produced using nucleotide analogs. It can be single-stranded or double-stranded. Such nucleic acids or polynucleotides include, but are not limited to, the coding sequences of structural genes, antisense sequences, and non-coding regulatory sequences that do not encode an mRNA or protein product. These terms also include genes. The terms "gene", "allele", or "gene sequence" are used broadly to refer to DNA nucleic acids associated with a biological function. Thus, a gene can include introns and exons in a genomic sequence, or can contain only the coding sequence in cDNA, and / or can include cDNA in combination with regulatory sequences. Thus, according to various aspects of the present invention, genomic DNA, cDNA, or coding DNA can be used. In one aspect, the nucleic acid is cDNA or coding DNA. The terms "peptide", "polypeptide", and "protein" are used interchangeably herein to refer to a polymer form of amino acids of any length joined together by peptide bonds. The term "allele" refers to any one of one or more alternative forms of a gene at a particular locus. A heterozygous allele is two different alleles at the same locus. A homozygous allele is two identical alleles at a particular locus. A wild-type (wt) allele is a naturally occurring allele without modification at the target locus.

[0075] It is shown for the first time herein that disrupting the NF2 gene, which encodes the Merlin protein, a key regulator of multiple pathways involved in cell proliferation, can accelerate the doubling time of porcine and bovine cells with different cell types or genetic backgrounds when transferred from adherent to suspension conditions. This allows the cells to be used in cell agriculture, where cells must proliferate rapidly under suspension conditions. We also show that disrupting the NF2 gene accelerates the doubling time faster than other genes in the Hippo pathway, suggesting that the effect of NF2 may be the result of its involvement in pathways other than the Hippo pathway.

[0076] The expression of the NF2 gene or the activity of the Merlin protein can be targeted in a variety of different ways, including at least one of the following:

[0077] 1) Modification at the gene level by:

[0078] a. Knocking out or reducing the activity / transcription / translation level by editing the coding sequence, promoter, intron, regulatory region;

[0079] b. RNA-guided DNA methylation; or

[0080] c. Transcriptional activation or repression using CRISPRa or CRISPRi or similar target-specific methods;

[0081] d. Knocking out or reducing the activity / transcription / translation level by non-directed means such as radiation or chemical mutagenesis;

[0082] 2) Modifications at the post-transcriptional level (post-transcriptional gene silencing) are carried out by the following methods:

[0083] a. RNAi or siRNA to reduce the translation of mRNA into protein; or

[0084] b. Site-specific nucleases to modify or cleave mRNA, such as CRISPR / Cas13a;

[0085] 3) Modifications at the post-translational level (protein destruction) are carried out by the following methods:

[0086] a. Comprising an activity-blocking / reducing molecule, wherein the activity-blocking / reducing molecule is a small molecule, an antibody, etc.; or

[0087] b. Comprising a protein degradation component, wherein the protein degradation component is a specialized protease, exoprotease or endoprotease.

[0088] In one embodiment, the expression of the NF2 gene can be modified to knock out the expression of the Merlin protein. In a related embodiment, CRISPR can be used for knocking out the expression of the NF2 gene. In another embodiment, single-point mutations or multi-point mutations can be used to knock out the expression of the NF2 gene. In still another embodiment, the CRISPR / Cas9 or CRISPR / Cas13a system can be used to knock out the expression of our NF2 gene.

[0089] According to one embodiment, the expression of the NF2 gene or the activity of the Merlin protein can be modified in animal cells by at least one small molecule or at least one RNAi. In one embodiment, the RNAi can be shRNA or siRNA.

[0090] When double-stranded RNA is processed by an RNase III-like protein called Dicer, mRNA within the cell can be silenced, reducing or eliminating protein activity. Dicer typically contains an N-terminal RNA helicase domain, an RNA-binding so-called Piwi / Argonaute / Zwille (PAZ) domain, two RNase III domains, and a double-stranded RNA-binding domain (dsRBD) (Collins et al., 2005). Dicer processing of long double-stranded RNA produces double-stranded siRNAs of 21-24 nucleotides, with a 2-base 3’ overhang and 5’ phosphate and 3’ hydroxyl groups. The resulting siRNA duplexes are then incorporated into an effector complex called the RNA-induced silencing complex (RISC), where the antisense or guide strand of the siRNA guides RISC to recognize and cleave the target mRNA sequence (Elbashir et al., 2001) when the double-stranded siRNA molecule is ATP-dependently unwound by RNA helicase activity (Nykanen et al., 2001). The catalytic activity of RISC results in mRNA degradation, mediated by the endonuclease Argonaute 2 (AGO2) (Liu et al., 2004; Song et al., 2004). AGO2 belongs to the highly conserved Argonaute protein family. Argonaute proteins are ~100 kDa highly basic proteins that contain two common domains, namely the PIWI and PAZ domains (Cerutti et al., 2000). The PIWI domain is crucial for interaction with Dicer and contains the nuclease activity responsible for cleaving mRNA. AGO2 uses one strand of the siRNA duplex as a guide to seek messenger RNAs containing complementary sequences and cleaves the phosphodiester backbone between bases 10 and 11 relative to the 5’ end of the guide strand (Elbashir et al., 2001). An important step in the RISC activation process is the cleavage of the sense or passenger strand by AGO2, removing that strand from the complex (Rand et al., 2005). Once the mRNA is cleaved, due to the presence of unprotected RNA ends in the fragments, the mRNA is further cleaved and degraded by intracellular nucleases and is no longer translated into protein. This results in a reduction in specific mRNA molecules and the corresponding proteins. This natural gene silencing mechanism can be exploited to regulate any selected one or more genes.

[0091] Numerous studies have been published describing how to optimize siRNAs, such as WO02 / 44321 (Walton SP et al., 2010; Chang Cl et al., 2011), the content of which is incorporated herein by reference.

[0092] Godinho and Khvorova 2019 describe common methods for delivering RNAi into cells. Examples of materials used as non-viral vectors, nanocarriers or in nanosystems for formulation and ligands for conjugation are described. Other examples are provided in the references incorporated herein. Nanoparticles are commonly used to introduce RNAi into cells, and cationic lipids (such as D-Lin-MC3-DMA), polymers (such as cyclodextrin-based polymers and biocollagen), polypeptides and exosomes are all examples of biomaterials that can be used to transport RNAi into the cytoplasm of target cells.

[0093] The generation and delivery of RNAi are further described in US6,506,559 and WO2007045930, the contents of which are incorporated herein by reference.

[0094] In some embodiments, post-translational inactivation (protein disruption) can be used to inhibit the activity of the Merlin protein. In one embodiment, an activity-blocking / reducing molecule can inhibit Merlin protein activity. For example, small molecules can inhibit Merlin protein activity.

[0095] In some embodiments, the Merlin protein can be degraded using protein degradation components. Examples include i) specialized proteases, such as calcium-dependent cysteine proteases, such as calpain, ii) exoproteases, or iii) endoproteases.

[0096] According to various aspects of the present invention, the modification can be in the promoter region or coding region of one or more targeted genes. Thus, the cell is genetically engineered / modified.

[0097] In one embodiment of an aspect of the present invention, the modified cell is a primary cell. In another embodiment of an aspect of the present invention, the modified cell is a somatic cell. Any somatic cell suitable for cell agriculture, i.e., food derived from animals produced by cell culture, is within the scope of the present invention. For example, the cell can be an adipocyte or a muscle cell. For example, the cell can be selected from one or more of the following cell types: myoblasts, fibroblasts, myofibroblasts, adipose-derived stem cells, epithelial cells, mesenchymal stem cells, satellite cells, iPSCs or hepatocytes.

[0098] The terms "animal" and "non-human animal" with respect to an animal and cells derived from said animal are used interchangeably herein and refer only to cells of non-human animals. The cells used in the present invention can be of any other animal origin. However, these cells are not human cells. Cells suitable for cell agriculture are preferably non-human animal cells that provide any source of dietary protein, fat and / or carbohydrate.

[0099] These cells are cells of non-human animals that are suitable for human and animal consumption. These animals include, for example, non-human mammals, birds, fish, crustaceans, mollusks, reptiles, amphibians, or insects. Exemplary non-human mammals include mammals of the subfamily Bovinae, Camelidae, Canidae, Caprae, Cervidae, Felidae, Equidae, Lagomorpha, Macropodidae, Oves, rodents, or Suidae. The cells can be cells of any livestock or poultry. The cells can be pigs, bovine animals (such as cows), sheep, goats, avine birds, or fish. The cells can be shrimp, prawns, crabs, crayfish, and / or lobsters. In one embodiment, the animal is a pig or a bovine animal (such as a cow).

[0100] The animals used in various aspects of the present invention can be animal species used in agriculture. The animal species used are animals raised for humans. These animals are listed above. In a preferred embodiment, they include pigs, bovine animals (such as cows), poultry (such as chickens, turkeys, ducks, geese), sheep, goats, equine animals, camelid animals, fish, crustaceans, or mollusks.

[0101] The doubling time of a cell line refers to the average time required for the size of a cell population to double due to cell cycle progression and subsequent division. Thus, removing cell cycle checkpoint inhibition in the cell cycle can reduce the time required for a cell to undergo mitosis and form two new daughter cells. When applied to the entire cell population of a cell line, this modification shortens the doubling time of the cell line, meaning that the cells expand rapidly and are more suitable for use in cell agriculture.

[0102] The term "gene modification" refers to a modification that alters the expression of a targeted gene or the functional activity of a gene product, i.e., the NF2 gene encoding the Merlin protein. Gene modification can result in loss of function, for example, by generating a knockout. To generate a loss-of-function / knockout, a mutation can be introduced into the coding sequence to render the expressed protein non-functional (such as amino acid substitution, deletion, or addition / insertion), or to generate a premature stop codon / to prevent the expression of a functional protein.

[0103] Examples of loss-of-function mutations are described herein. However, as described herein, any mutation that results in a dominant loss of function is included within the scope of the present invention. As used herein, "dominant" also includes "semi-dominant" or "partially dominant". Thus, the mutant allele can be fully dominant, partially dominant, or semi-dominant. Preferably, the mutant allele is fully dominant. Loss-of-function mutations include knockout modifications or any other modification that causes an amino acid substitution or alteration, where the substitution or alteration results in the resulting protein lacking a specific function or in a decrease in the activity of the protein or in preventing the expression of the protein. Preferably, both alleles of NF2 are knocked out.

[0104] A knockout modification or mutation can at least partially eliminate a specific endogenous nucleic acid sequence from the genomic DNA of a cell encoding a target protein. By eliminating the corresponding nucleic acid sequence, the protein can no longer be synthesized by the cellular machinery. In some embodiments, the knockout modification occurs by introducing an insertion / deletion (indel) (insertion and / or deletion event), resulting in a change in the native amino acid composition of the resulting protein. This typically occurs in the form of a frameshift mutation.

[0105] Examples of gain of function are also described herein. In some embodiments, a change in the gene sequence may result in a change in the native amino acid composition of the resulting protein, which increases the activity of the protein. In additional embodiments, a change in the amino acid sequence of the protein may add or remove regulatory regions of the protein, such as, but not limited to, phosphorylation, acetylation, and ubiquitination sites. In additional embodiments, a change in the amino acid sequence of the protein may stabilize or destabilize intermediate states of its enzymatic reaction substrates and products. In additional embodiments, a change in the amino acid sequence of the protein may lock it in a permanent "on" state. In additional embodiments, a change in the regulatory gene sequence may increase the transcription of the gene, thereby increasing the amount of protein per cell and the total activity of the protein. In additional embodiments, a change in the amino acid sequence of the protein may affect its interaction with other proteins. In additional embodiments, a gain of function alteration is introduced in a targeted manner by knocking in specific nucleotides that result in the desired amino acid change at the protein level or the desired nucleotide exchange in the regulatory gene region. In additional embodiments, a gain of function mutation is introduced into the cell by adding an exogenous nucleotide sequence encoding the desired protein.

[0106] Amino acid substitutions are affected by changes in the nucleic acid sequence that result in a different amino acid at a given site. Such modifications may or may not affect the functional properties and / or activity of the encoded polypeptide. Conservative substitutions are well known in the art. For example, the codon for the amino acid alanine (a hydrophobic amino acid) can be replaced by a codon encoding another less hydrophobic residue (e.g., glycine) or a more hydrophobic residue (e.g., valine, leucine, or isoleucine). Similarly, changes that result in the replacement of one residue with a negatively charged residue (e.g., replacing glutamate with aspartate) or one residue with a positively charged residue (e.g., replacing arginine with lysine) can also be expected to produce functionally equivalent products. Nucleotide changes that result in alterations in the N-terminal and C-terminal portions of the polypeptide molecule are also not expected to alter the activity of the polypeptide. Each of the proposed modifications is well within the routine skill in the art, as is the determination of the retention of the biological activity of the encoded product. Non-conservative substitutions result in an encoded protein that does not retain the same functional properties and / or activity as the unmodified protein.

[0107] Sequence identity is generally defined with reference to the algorithm GAP (Wisconsin GCG software package, Acceleys Inc, San Diego, USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences, maximizing the number of matches and minimizing the number of gaps. Typically, with default parameters, the gap creation penalty is usually equal to 12 and the gap extension penalty is equal to 4. The use of GAP may be preferred, but other algorithms such as BLAST, or the Smith-Waterman algorithm or the TBLASTN program, usually with default parameters, can also be used. In particular, the psi-Blast algorithm can be used. Sequence identity can be defined using Bioedit, the ClustalW algorithm. The alignment can be performed using Snapgene and based on the MUCLE (Multiple Sequence Comparison by Log-Expectation) algorithm. Sanger sequencing of PCR amplicons and analysis of the sequencing results using the ICE analysis tool (ICE.synthego.com) are also used to confirm sequence identity.

[0108] In one embodiment, targeted genomic modification and / or rare-cutting endonucleases such as TALENs, ZFNs or CRISPR / Cas9 are used to introduce modifications.

[0109] Genome editing technologies have become alternatives to traditional mutagenesis methods such as physical and chemical mutagenesis, or methods using transgenic expression in animal cells to generate mutant animal cells with improved phenotypes, which are important in cell research and cell agriculture. These technologies employ sequence-specific nucleases (SSNs), including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and RNA-guided nuclease Cas9 (CRISPR / Cas9), which create targeted DNA double-strand breaks (DSBs) that are then repaired mainly by error-prone non-homologous end joining (NHEJ) or high-fidelity homologous recombination (HR).

[0110] As explained in detail below, targeted genomic modification based on such editing techniques can be used to introduce mutations according to various aspects of the present invention into animal cells.

[0111] In another aspect, the present invention also relates to a method of modifying the expression or function of one or more genes in a non-human animal, wherein the genes are related to genome surveillance, cell cycle control and / or cell death control. In a preferred embodiment, the animal is an animal suitable for human or animal consumption, such as for agriculture. In an embodiment, the method comprises introducing a mutation into one or more genes of an animal cell.

[0112] In another aspect, the present invention relates to a method for producing the modified non-human animal cells described herein, wherein the method comprises introducing a genetic modification in one or more genes related to genomic surveillance, cell cycle control, and / or cell death control. In a preferred embodiment, the animal is an animal for agriculture. The method is carried out in vitro or ex vivo.

[0113] In yet another aspect, the present invention provides modified animal cells that have a genetic modification in the NF2 gene. In one embodiment, the modified animal cells can be immortalized animal cells. In additional embodiments, the immortalized animal cells may have been immortalized using targeted genomic modification. In other embodiments, the immortalized animal cells may have been immortalized using random mutagenesis. In still further embodiments, the immortalized animal cells may have been immortalized using radiation or chemical mutagenesis. In still further embodiments, the immortalized animal cells may have been spontaneously immortalized.

[0114] In some embodiments, the genetic modification in the NF2 gene can be created using targeted genomic modification. In other embodiments, the genetic modification in the NF2 gene can be created using random mutagenesis. In still further embodiments, the genetic modification in the NF2 gene may have occurred spontaneously.

[0115] In all aspects of the present invention, the animal is not a human. Animals that can be used are listed herein, particularly animals related to agriculture.

[0116] Targeted genome modification in animal cells using gene editing

[0117] Targeted genome modification or targeted genome editing is a genome engineering technique that uses targeted DNA double-strand breaks (DSBs) to facilitate genome editing through homologous recombination (HR)-mediated recombination events. To achieve efficient genome editing by introducing site-specific DNA DSBs, four main classes of customizable DNA-binding proteins can be used: meganucleases derived from microbial mobile genetic elements, ZF nucleases based on eukaryotic transcription factors, rare-cutting endonucleases / sequence-specific nucleases (SSNs) (e.g., TALENs, transcription activator-like effectors (TALEs) from Xanthomonas), and the RNA-guided DNA endonuclease Cas9 from the type II bacterial adaptive immune system CRISPR (clustered regularly interspaced short palindromic repeats). Other CRISPR / Cas systems using different Cas proteins are well known to those skilled in the art. Meganucleases, ZF, and TALE proteins all recognize specific DNA sequences through protein-DNA interactions. Although meganucleases integrate their nuclease and DNA-binding domains, ZF and TALE proteins are each composed of individual modules that target 3 or 1 nucleotide (nt) of DNA. ZFs and TALEs can be assembled in the desired combination and attached to the nuclease domain of FokI to direct nuclease activity to specific genomic sites.

[0118] After being delivered into host cells through the bacterial type III secretion system, TAL effectors enter the nucleus, bind to effector-specific sequences in the host gene promoter, and activate transcription. Their targeting specificity is determined by a central domain of tandem 33-35 amino acid repeats. This is followed by a single truncated repeat of 20 amino acids. Most of the naturally occurring TAL effectors tested have 12 to 27 complete repeats.

[0119] These repeat sequences differ only in two adjacent amino acids, i.e., their repeat variable di-residues (RVDs). The RVD determines which single nucleotide the TAL effector will recognize: one RVD corresponds to one nucleotide, and the four most common RVDs each preferentially bind to one of the four bases. Naturally occurring recognition sites are preceded by a T required for TAL effector activity. TAL effectors can be fused to the catalytic domain of the Fokl nuclease to create TAL effector nucleases (TALENs), which generate targeted DNA double-strand breaks (DSBs) in vivo for genome editing. The use of this technology in genome editing is well described in the art, e.g., in US 8,440,431, US 8,440,432, and US 8,450,471. Custom plasmids can be used with the Golden Gate cloning method to assemble multiple DNA fragments. The Golden Gate method uses type IIS restriction endonucleases, which cut outside the recognition site, generating unique 4bp overhangs. Cloning is accelerated by digesting and ligating in the same reaction mixture because correct assembly clears the enzyme recognition sites. The assembly of custom TALEN or TAL effector constructs involves two steps: (i) assembling the repeat modules into an intermediate array of 1-10 repeats, and (ii) ligating the intermediate array into a backbone to form the final construct.

[0120] Another genome editing method that can be used according to various aspects of the present invention is CRISPR. The use of this technology in genome editing is well described in the art, e.g., in US 8,697,359. Briefly, CRISPR is a microbial nuclease system involved in the defense against invading phages and plasmids. The CRISPR locus in a microbial host contains a combination of CRISPR-associated (Cas) genes and non-coding RNA elements capable of programming the specificity of CRISPR-mediated nucleic acid cleavage. Three types (I-III) of CRISPR systems have been identified in a wide range of bacterial hosts. A key feature of each CRISPR locus is the presence of a series of repeat sequences (direct repeats), which are separated by short segments of non-repeating sequences (spacers). The non-coding CRISPR array is transcribed and cleaved into short crRNAs containing a single spacer sequence, which guides the Cas nuclease to the target site (protospacer).

[0121] "crRNA" or CRISPR RNA refers to an RNA sequence containing a protospacer element and additional nucleotides complementary to tracrRNA. "tracrRNA" (trans-activating RNA) refers to an RNA sequence that hybridizes with crRNA and binds to a CRISPR enzyme (such as Cas9), thereby activating the nuclease complex to introduce a double-strand break at a specific site in the genomic sequence of at least one nucleic acid or the promoter sequence of one or more genes. "Protospacer element" refers to the part of crRNA (or sgRNA) that is complementary to the genomic DNA target sequence, usually about 20 nucleotides in length. This can also be referred to as the spacer or targeting sequence.

[0122] "sgRNA" (single guide RNA) refers to the combination of tracrRNA and crRNA in a single RNA molecule, preferably also including a linker loop (which connects tracrRNA and crRNA into a single molecule). "sgRNA" can also be referred to as "gRNA", and in the current context, these terms are interchangeable. sgRNA or gRNA provides targeting specificity and scaffold / binding ability for Cas nucleases. gRNA can refer to a double RNA molecule containing a crRNA molecule and a tracrRNA molecule.

[0123] Type II CRISPR is one of the best-characterized systems and makes targeted double-strand breaks in DNA in four sequential steps. First, two non-coding RNAs, the pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus. Second, tracrRNA hybridizes to the repeat sequence regions of the pre-crRNA and mediates the processing of the pre-crRNA into mature crRNAs containing individual spacer sequences. Third, the mature crRNA:tracrRNA complex guides Cas9 to the target DNA via Watson-Crick base pairing between the spacer on the crRNA and the protospacer on the target DNA next to the protospacer adjacent motif (PAM), which is an additional requirement for target recognition. Finally, Cas9 mediates cleavage of the target DNA to generate a double-strand break within the protospacer. Thus, Cas9 is the signature protein of the type I CRISPR-Cas system, a large monomeric DNA nuclease that is guided to DNA target sequences adjacent to PAM sequence motifs by a complex of two non-coding RNAs: CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The Cas9 protein contains two nuclease domains homologous to RuvC and HNH nucleases. The HNH nuclease domain cleaves the complementary DNA strand, while the RuvC-like domain cleaves the non-complementary strand, introducing a blunt cut in the target DNA. Heterologous expression of Cas9 together with a guide RNA (gRNA) (also known as single-guide RNA (sgRNA)) can introduce site-specific double-strand breaks (DSBs) into the genomic DNA of living cells of various organisms. For applications in eukaryotes, a codon-optimized version of Cas9, originally from the bacterium Streptococcus pyogenes, has been used.

[0124] Synthetic CRISPR systems typically consist of two components, a gRNA and a non-specific CRISPR-associated endonuclease, and can be used to generate knockout cells or animals by co-expressing a gRNA specific for the gene to be targeted and capable of associating with the endonuclease Cas9. Notably, the gRNA is an artificial molecule that contains a domain that interacts with Cas or any other CRISPR effector protein or its variant or catalytically active fragment, as well as another domain that interacts with the target nucleic acid, and thus represents a synthetic fusion of crRNA and tracrRNA. The genomic target can be any 20-nucleotide DNA sequence as long as the target is located immediately upstream of a PAM sequence. The PAM sequence is important for target binding, and the exact sequence depends on the type of Cas9.

[0125] The PAM sequences for Cas9 from Streptococcus pyogenes are described as "NGG" or "NAG" (standard IUPAC nucleotide code) (Jinek et al., "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity", Science 2012, 337:816-821). The PAM sequences for Cas9 from Staphylococcus aureus are "NNGRRT" or "NNGRR(N)". Other variant CRISPR / Cas9 systems are known to exist. Thus, Neisseria meningitidis Cas9 cuts at the PAM sequence NNNNGATT. Streptococcus thermophilus Cas9 cuts at the PAM sequence NNAGAAW. More recently, additional PAM motifs of the Campylobacter CRISPR system, NNNNRYAC, have been described (WO 2016 / 021973). For the Cpf1 nuclease, such as Cas12a, it has been described that the Cpf1-crRNA complex effectively recognizes and cuts target DNA after a short T-rich PAM in the absence of tracrRNA, which is different from the common G-rich PAMs recognized by the Cas9 system. In addition, by using modified CRISPR polypeptides, specific single-strand breaks can be obtained. The combined use of Cas nickases with various recombinant gRNAs can also induce highly specific DNA double-strand breaks by a dual DNA nicking approach. Furthermore, by using two gRNAs, the specificity of DNA binding can be optimized, and thus DNA cleavage can be optimized. Meanwhile, there are other CRISPR effectors, such as the CasX and CasY effectors originally described for bacteria, which are also available and represent additional effectors that can be used for genome engineering purposes (Burstein et al., "New CRISPR-Cas systems from uncultivated microbes", Nature, 2017, 542, 237-241).

[0126] Once expressed, the Cas9 protein and gRNA form a ribonucleoprotein complex through the interaction between the gRNA "scaffold" domain and a surface-exposed positively charged groove on Cas9. Cas9 undergoes a conformational change upon gRNA binding, converting the molecule from an inactive, non-DNA-binding conformation to an active DNA-binding conformation. Importantly, the "spacer" sequence of the gRNA remains free to interact with the target DNA. The Cas9-gRNA complex will bind to any genomic sequence with a PAM, but the degree of match between the gRNA spacer and the target DNA determines whether Cas9 will cleave. Once the Cas9-gRNA complex binds to a putative DNA target, the "seed" sequence at the 3' end of the gRNA targeting sequence begins to anneal to the target DNA. If the seed and target DNA sequences match, the gRNA will continue to anneal to the target DNA in the 3' to 5' direction (relative to the polarity of the gRNA).

[0127] When properly designed, CRISPR / Cas9 and similar CRISPR / Cpf1 and other CRISPR systems are highly specific, but particularly, specificity remains a major issue, especially for clinical applications based on CRISPR technology. The specificity of the CRISPR system depends largely on the degree of specificity of the gRNA targeting sequence for the genomic target compared to other parts of the genome. The sgRNA is a synthetic RNA chimera produced by fusing the crRNA with the tracrRNA Combined. Located The sgRNA guide sequence at its end confers DNA target specificity. Therefore, by modifying the guide sequence, sgRNAs with different target specificities can be created. The typical length of the guide sequence is 20 bp.

[0128] Thus, as used herein, the term "guide RNA" refers to the synthetic fusion of two RNA molecules, namely the crRNA (CRISPR RNA) containing a variable targeting domain and the tracrRNA. In one embodiment, the guide RNA comprises a variable targeting domain of 12 to 30 nucleotide sequences and an RNA fragment that can interact with the Cas endonuclease.

[0129] Described below are sgRNAs applicable to the method of the present invention. As used herein, the term "guide polynucleotide" refers to a polynucleotide sequence that can form a complex with a Cas endonuclease and enable the Cas endonuclease to recognize and optionally cleave a DNA target site. The guide polynucleotide can be a single molecule or a double molecule. The guide polynucleotide sequence can be an RNA sequence, a DNA sequence, or a combination thereof (RNA-DNA combined sequence). Optionally, the guide polynucleotide can comprise at least one nucleotide, phosphodiester bond, or linkage modification, such as but not limited to locked nucleic acid (LNA), 5-methyl dC, 2,6-diaminopurine, 2'-Fluoro A, 2'-Fluoro U, 2'-O-methyl RNA, phosphorothioate bond, linkage with a cholesterol molecule, linkage with a polyethylene glycol molecule, linkage with a spacer 18 (hexaethylene glycol chain) molecule, or a 5' to 3' covalent linkage resulting in cyclization. Guide polynucleotides consisting solely of ribonucleic acids are also contemplated.

[0130] The terms "target site", "target sequence", "target DNA", "target locus", "genomic target site", "genomic target sequence", and "genomic target locus" are used interchangeably herein and refer to such a polynucleotide sequence in the cell genome (including chloroplast and mitochondrial DNA) in which a Cas endonuclease induces a double-strand break in the cell genome. The target site can be an endogenous site in the genome, or alternatively, the target site can be heterologous to the plant and thus not naturally present in the genome, or the target site can be found at a heterologous genomic location compared to the naturally occurring site. As used herein, the terms "endogenous target sequence" and "native target sequence" are used interchangeably herein and refer to a target sequence that is endogenous or native to the genome and is located at the endogenous or native position of the target sequence in the genome.

[0131] The length of the target site can vary and includes, for example, target sites having a length of at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotides. It is also possible that the target site can be palindromic, that is, the sequence on one strand reads the same in the opposite direction as the complementary strand. The nick / cut site can be within the target sequence, or the nick / cut site can be outside the target sequence. In another variant, the cleavage can occur at nucleotide positions directly opposite each other to produce a blunt end cut, or in other cases, the incision can be staggered to produce single-stranded overhangs, also referred to as "sticky ends", which can be 5' overhangs or 3' overhangs.

[0132] In one embodiment, the Cas endonuclease gene is a Cas9 endonuclease, such as, but not limited to, the Cas9 genes listed in WO2007 / 025097 incorporated herein by reference. In another embodiment, the Cas endonuclease gene is an animal-optimized Cas9 endonuclease.

[0133] In one embodiment, the Cas endonuclease gene is a Streptococcus pyogenes Cas9 gene optimized for animal codons, which can recognize any genomic sequence of the form N(12 - 30)NGG that can in principle be targeted.

[0134] In one embodiment, the Cas endonuclease is introduced directly into the cell by any method known in the art, such methods including, but not limited to, transient introduction methods, transfection, and / or topical application.

[0135] The Cas9 expression plasmid for use in the methods of the present invention can be constructed as described in the art.

[0136] In one embodiment, the targeted genomic modification according to the various aspects of the present invention includes the use of rare-cutting endonucleases, such as TALENs, ZFNs, or CRISPR / Cas; for example, the use of CRISPR / Cas9. Rare-cutting endonucleases / sequence-specific endonucleases are natural or engineered proteins with endonuclease activity and are target-specific. These endonucleases bind to nucleic acid target sequences with recognition sequences typically 12 - 40 bp in length. In one embodiment, the SSN is selected from TALENs. In another embodiment, the SSN is selected from CRISPR / Cas9. This is described in more detail below.

[0137] In one embodiment, the step of introducing a mutation includes contacting a population of animal cells with a DNA-binding protein that targets the NF2 gene. In one embodiment, the method includes contacting the cell population with one or more rare-cutting endonucleases, such as ZFNs, TALENs, or CRISPR / Cas9 that target the NF2 gene.

[0138] The method may further include the steps of selecting cells in which the NF2 gene sequence has been modified from the population and regenerating the selected animal cells.

[0139] In an embodiment, the method includes the use of CRISPR / Cas9. Thus, in this embodiment, the method includes introducing and co-expressing Cas9 and an sgRNA targeting the NF2 gene sequence in animal cells, and screening for induced targeted mutations in the NF2 gene.

[0140] Cas9 and sgRNA can be included in a single or two expression vectors. The target sequence is the NF2 nucleic acid sequence as shown herein.

[0141] In one embodiment, screening for CRISPR-induced targeted mutations in the NF2 gene comprises obtaining a DNA sample from transfected / transduced animal cells and performing DNA amplification and optionally restriction enzyme digestion to detect mutations in the NF2 gene.

[0142] In one embodiment, the restriction enzyme is the mismatch-sensitive T7 endonuclease. T7E1 is an enzyme specific for heteroduplex DNA caused by genome editing.

[0143] The PCR fragments amplified from the transfected / transduced animal cells are then evaluated using a gel electrophoresis-based assay. In a further step, the presence of the mutation can be confirmed by sequencing the NF2 gene. Genomic DNA (i.e., wt and mutant) can be prepared from each sample and the DNA fragment containing each target site amplified by PCR. The PCR products are digested with a restriction enzyme as the target locus includes a restriction enzyme site. The restriction enzyme site is disrupted by CRISPR- or TALEN-induced mutations by NHEJ or HR, so the mutant amplicons are resistant to restriction enzyme digestion and result in uncut bands. Alternatively, the PCR products are digested with T7E1 (the T7E1 enzyme generates cleaved DNA specific for heteroduplex DNA caused by genome editing) and visualized by agarose gel electrophoresis. In a further step, they are sequenced.

[0144] In one embodiment, the method uses an sgRNA (and template, synthetic single-stranded DNA oligonucleotide (ssDNA oligonucleotide), or donor DNA) construct, as defined in detail below, to introduce a targeted SNP or mutation, particularly one of the substitutions described herein, into the NF2 gene and / or promoter. The template DNA strand is introduced after the sgRNA-mediated SNP in double-stranded DNA and can be used to generate a specific targeted mutation (i.e., SNP) in the gene using homology-directed repair. Synthetic single-stranded DNA oligonucleotides (ssDNA oligonucleotides) or DNA plasmid donor templates can be used for precise genome modification via the homology-directed repair (HDR) pathway. Homologous recombination is the exchange of DNA sequence information by using sequence homology. Homology-directed repair (HDR) is a homologous recombination process in which a DNA template is used to provide the homology required for precise repair of double-strand breaks (DSBs). The CRISPR guide RNA programs the Cas9 nuclease to cut genomic DNA at specific locations. Once a double-strand break (DSB) occurs, mammalian cells utilize endogenous mechanisms to repair the DSB. In the presence of donor DNA (ssDNA oligonucleotide or plasmid donor), HDR can be used to precisely repair the DSB, thereby generating the desired genomic alterations (insertions, deletions, or replacements).

[0145] Single-stranded DNA donor oligonucleotides are delivered into cells to insert or alter short DNA sequences (SNPs, amino acid substitutions, epitope tags, etc.) in the endogenous genomic target region.

[0146] A "donor sequence" is a nucleic acid sequence that contains all the elements required to introduce a specific substitution into a target sequence, preferably using homology-directed repair (HDR). In one embodiment, the donor sequence includes a repair template sequence for introducing at least one SNP. Preferably, the repair template sequence is flanked by at least one arm, preferably a left arm and a right arm, more preferably each arm being about 100 bp and identical to the target sequence. More preferably, one or more arms flank two gRNA target sequences containing PAM motifs such that the donor sequence can be released by Cas9 / gRNAs.

[0147] The above method uses such animal cells in which an expression vector containing a sequence-specific nuclease has been introduced into the animal cells to target the NF2 nucleic acid sequence. The terms "introduce" or "transfect / transduce" as referred to herein include the transfer of exogenous polynucleotides into a host cell, regardless of the method used for the transfer.

[0148] Advantageously, any one of several transfection / transduction methods can be used to introduce the target gene into a suitable cell. The methods for transfection / transduction of animal cells can be used for transient or stable transfection / transduction. Transfection / transduction methods include the use of liposomes, electroporation, chemicals that increase the uptake of free DNA, direct injection of DNA into animal cells, particle bombardment as described in the examples, transfection / transduction using viruses, or microinjection. The methods can be selected from microinjection into animal material, particle bombardment with DNA- or RNA-coated particles, infection (non-integrating or integrating) with viruses, etc.

[0149] After DNA transfer and regeneration, putatively transformed animal cells can also be evaluated, for example using Southern analysis, to determine the presence of the target gene, copy number, and / or genomic organization. Alternatively or additionally, Northern and / or Western analysis can be used to monitor the expression level of the newly introduced DNA, both of which techniques are well known to those of ordinary skill in the art.

[0150] Sequence-specific nucleases can also be introduced into animal cells as part of an expression vector. The vector can contain one or more replication systems that enable it to replicate in the host cell. Self-replicating vectors include plasmids, cosmids, and viral vectors. Alternatively, the vector can be an integrating vector that allows the DNA sequence to integrate into the chromosome of the host cell. The vector ideally also has unique restriction sites for inserting DNA sequences. If the vector does not have unique restriction sites, it can be modified to introduce or eliminate restriction sites to make it more suitable for further manipulation. Vectors suitable for expressing nucleic acids are known to those of skill in the art, non-limiting examples being pcDNA3.1. The nucleic acid is inserted into the vector such that it is operably linked to a suitable animal-active promoter. Suitable animal-active promoters for use with nucleic acids include, but are not limited to, PGK, CMV, EF1a, CAG, SV40, and Ubc.

[0151] In an embodiment of the invention, the promoter region or coding region of one or more genes is modified.

[0152] In one embodiment, the cultured animal cells further comprise at least one additional genetic modification to manipulate genomic surveillance, cell cycle control, and / or cell death control pathways. Genetic modification of the NF2 gene or modification of Merlin protein activity can be used in combination with other genetic modifications to further help reduce the doubling time. See, for example, the applicant's own patent application PCT / GB2023 / 052528, which is incorporated herein by reference. Thus, in one embodiment, the at least one additional genetic modification can be in one or more of the following genes: RB1, TP53, and / or RAS genes.

[0153] Genetic modifications of the RB1, TP53, and / or RAS genes can be carried out alone or in combination with genetic modifications in the NF2 gene in any combination. In one embodiment, the genetic modification can be a single-point mutation or a multi-point mutation to knockout gene expression for, for example, the RB1 and TP53 genes, or the genetic modification can be a single-point mutation or a multi-point mutation to knockout gene expression for, for example, the RAS gene.

[0154] In one embodiment, the animal cell has an additional genetic modification in RB1. In one embodiment, the animal cell has an additional genetic modification in TP53. In one embodiment, the animal cell has an additional genetic modification in the RAS gene. In one embodiment, the animal cell has additional genetic modifications in RB1 and TP53. In one embodiment, the animal cell has additional genetic modifications in RB1 and the RAS gene. In one embodiment, the animal cell has additional genetic modifications in TP53 and the RAS gene. In one embodiment, the animal cell has additional genetic modifications in RB1, TP53, and the RAS gene. In one embodiment, the RAS gene is HRAS, NRAS, or KRAS. In one embodiment, the RAS gene is HRAS.

[0155] Suitable sequences from the genes of pigs (wild boars) (Sus-scrofa) are described below (see also Table 1). Thus, the modified cells can be porcine cells, and the targeted genes are selected from RB1, TP53, and / or HRAS, NRAS, or KRAS.

[0156] For example, in one embodiment, exon 2 of porcine HRAS (wild-type sequence shown in SEQ ID No: 31) and the modified cells can contain modifications in porcine HRAS exon 2, as shown in SEQ ID No: 32, 33, and / or 40. Mutations in HRAS can include Gly>Val (aa12) [GGA>GTA], and optionally a PAM-blocking mutation Gly>Val (aa15) [GGG>GtG].

[0157] In another embodiment, exon 8 of porcine RB1 (wild-type sequence SEQ ID NO: 34) can be targeted, and the modified porcine RB1 exon 8 sequence can be as shown in SEQ ID No: 35 and / or 36.

[0158] In yet another embodiment, exon 5 of porcine TP53 (wild-type sequence SEQ ID No: 37) can be targeted, and the modified cells can contain modifications in porcine TP53, as shown in SEQ ID No: 38 and / or 39.

[0159] Alternative sequences of porcine HRAS (SEQ ID NO. 41), KRAS (SEQ ID NO. 42 (isoform A) and 43 (isoform B)), and NRAS (SEQ ID NO. 44) are provided in Table 1 and can also be targeted.

[0160] Those skilled in the art will know of other suitable genetic modifications to these and other genes, as well as suitable genetic modifications to these and other genes in other species, such as bovine species. For example, see the applicant's own patent application PCT / GB2023 / 052528.

[0161] The invention is not limited to modified porcine cells. Those skilled in the art will know that, in order to manipulate other animal cells from animal species suitable for human or animal consumption, such as suitable for human consumption, such as suitable for animal consumption, such as for agriculture, equivalent orthologues, i.e., endogenous RB1, TP53, and / or HRAS genes specific to the targeted non-human animal species, are genetically modified. Suitable gene sequences can be identified from public databases. Those skilled in the art are also able to use standard methods in the art to identify homologues and orthologues, such as based on sequence identity to porcine sequences, to identify suitable sequences.

[0162] In a further embodiment of the present invention, the gene is RAS and the modification is a hyperactivated modification. In one embodiment, the modification generated in the RAS protein keeps it in a constant active state. In one embodiment, the activating modification reduces GTP hydrolysis. The RAS gene can be any one of HRAS, NRAS or KRAS (isoform A or isoform B). In a related embodiment of the present invention, the hyperactivated modification includes one or more amino acid substitutions in the protein. The RAS gene can be a porcine RAS gene. The RAS gene can be a bovine RAS gene. For example, in one embodiment, the one or more amino acid substitutions include substituting the glycine at position 12 of SEQ ID NO: 41, 42, 43 or 44. In one embodiment, the one or more amino acid substitutions include substituting the glycine at position 12 of SEQ ID NO: 41, 42, 43 or 44, wherein the one or more amino acids are selected from the list consisting of alanine, cysteine, aspartic acid, arginine, serine and valine. In one embodiment, the one or more amino acid substitutions include substituting the glycine at position 12 of SEQ ID NO: 41, 42, 43 or 44, wherein the one or more amino acids are selected from the group consisting of alanine, cysteine, aspartic acid, arginine, serine and valine. In one embodiment, the one or more amino acid substitutions include substituting the glycine at position 12 of SEQ ID NO: 41, 42, 43 or 44 with valine. In one embodiment, the one or more amino acid substitutions include substituting the glycine at position 13 of SEQ ID NO: 41, 42, 43 or 44. In one embodiment, the one or more amino acid substitutions include substituting the glycine at position 13 of SEQ ID NO: 41, 42, 43 or 44, wherein the one or more amino acids are selected from the list consisting of alanine, cysteine, aspartic acid, arginine, serine and valine. In one embodiment, the one or more amino acid substitutions include substituting the glycine at position 13 of SEQ ID NO: 41, 42, 43 or 44, wherein the one or more amino acids are selected from the group consisting of alanine, cysteine, aspartic acid, arginine, serine and valine. In one embodiment, the one or more amino acid substitutions include substituting the glycine at position 13 of SEQ ID NO: 41, 42, 43 or 44 with valine. In one embodiment, the one or more amino acid substitutions include substituting the glutamine at position 61 of SEQ ID NO: 41, 42, 43 or 44. In one embodiment, the one or more amino acid substitutions include substituting the glycine at position 61 of SEQ ID NO: 41, 42, 43 or 44, wherein the one or more amino acids are selected from the list consisting of glutamic acid, histidine, lysine, proline, leucine and arginine.In one embodiment, the one or more amino acid substitutions include substituting the glycine at position 61 of SEQ ID NO: 41, 42, 43, or 44, wherein the one or more amino acids are selected from the list consisting of glutamic acid, histidine, lysine, proline, and arginine. In still further related embodiments, the one or more amino acid substitutions include substituting the glycine at position 12 of SEQ ID NO: 31 with valine. In one embodiment, a gain-of-function mutation is introduced into the RAS gene using a targeted nuclease or a derivative thereof. In another embodiment, a gain-of-function version of the RAS protein is introduced into the cell as an exogenous nucleotide. In one embodiment, a gain-of-function mutation is introduced by random mutagenesis, which includes but is not limited to chemical mutagenesis. In one embodiment, the gain-of-function mutation is introduced by spontaneous mutation. In another embodiment, RAS activity is increased by interfering with other regulators of RAS activity.

[0163] Cultured meat products and methods

[0164] In another aspect, the present invention provides a method for producing cultured meat / cultured meat products / food, which includes culturing the modified cells according to any one of the foregoing embodiments of the present invention. In related embodiments, the method includes continuous or batch culturing of the modified cells.

[0165] The term "cultured meat" is used herein to describe meat grown from in vitro animal cell cultures, as distinct from meat from slaughtered animals. Other terms that can be used in the art to describe meat grown from in vitro animal cell cultures include cultured meat, cell-grown meat, clean meat, laboratory-grown meat, test-tube meat, in vitro meat, tube steak, synthetic meat, cell-cultured meat, cell-grown meat, tissue-engineered meat, engineered meat, artificial meat, and cultured meat. Phrases such as "cell-based meat", "slaughter-free cell-based meat", "in vitro-produced meat", "in vitro cell-based meat", "cultured meat", "slaughter-free cultured meat", "in vitro-produced cultured meat", "in vitro meat", "in vitro cultured meat", and other similar phrases can be used interchangeably herein to refer to meat produced in vitro starting from cells in culture, and methods that do not involve slaughtering an animal to directly obtain meat from that animal for dietary consumption. The modified cells of the present invention can be suitable for human and / or non-human consumption. In some embodiments, cell-based meat is suitable for animal consumption, such as domestic animals. Thus, the cell biomass herein supports the growth of "pet food", such as dog food, cat food, etc.

[0166] In one embodiment, the invention is a cultured animal cell in which the expression of the NF2 gene is modified or the activity of the Merlin protein is modified, and wherein the animal is an animal species suitable for human or animal consumption. In a further embodiment, the cultured animal cell is a cultured or cultivated meat cell suitable for human or animal consumption. In a further embodiment, the cultured animal cell is an animal cell cultured in vitro. In yet a further embodiment, the cultured animal cell is not a method of treating cancer. In yet a further embodiment, the invention does not relate to the treatment of cancer or a product or genetic modification for treating cancer.

[0167] Batch culture refers to culturing cells in a closed system, where the cells are cultured for a defined period of time or until a defined criterion is reached. Once this criterion or time is met, the culture is stopped, the cells are harvested, the system is emptied and cleaned, and prepared for a new culture. Nutrients and / or culture additives can be added at the start of the culture or during the culture process. Continuous culture refers to culturing cells in a system where, after a period of growth, the cells are continuously removed, or removed at specific time points, while there remains a cell population capable of continuing to grow and divide in the system. This process is repeated for a set period of time or indefinitely. Nutrients and / or culture additives are added periodically or continuously so that the cells present in the system always have optimal conditions for growth and division.

[0168] In another embodiment, the invention provides a cultivated animal tissue comprising modified cells according to any one of the preceding embodiments of the invention.

[0169] In another aspect, the invention provides the use of a modified animal cell according to any one of the preceding embodiments for cellular agriculture.

[0170] In another aspect, the invention provides a method for producing an immortalized cell line, the method comprising a method according to any one of the preceding aspects of the invention, wherein the immortalized cell line comprises a modification of the NF2 gene. The modification can be a loss-of-function modification. The loss-of-function modification can be a knockout of the NF2 gene. Such a cell line can be used for cellular agriculture.

[0171] In a further aspect, the invention provides a method for producing cultured meat products, comprising culturing one or more modified animal non-human cells or cell lines according to any one of the preceding embodiments and optionally forming the cells into tissue-like structures. In a related embodiment, the method comprises forming the cells into muscle tissue-like structures. In a further aspect, the invention provides cultured meat products for human or non-human consumption, comprising the modified cells or cell lines of the invention.

[0172] In certain embodiments, cultured meat products refer to products in which cells according to the present invention form products acceptable and / or suitable and / or appropriate for human consumption. The structure of the product may mimic or be designed to mimic the tissues of an animal species for human consumption. Cultured meat products may have a tissue-like structure. The tissue may be selected from one or more of the following: muscle, fat, heart, liver, kidney, and / or any tissue for human consumption.

[0173] The tissue-like structure according to the present invention is a structure that is similar to a specific animal tissue in terms of texture, taste, mouthfeel, visual structure, visual texture, and color. The tissue-like structure does not have to be able to perform the body functions that the tissue performs in vivo. The tissue-like structure means that the tissue-like structure looks similar or identical to the tissue collected from an animal for consumption by consumers of cultured meat products.

[0174] Cultured meat products contain modified cells according to the present invention, but may additionally contain other ingredients, such as colorants, flavorings, and / or flavor enhancing compositions, as well as dietary supplements, such as vitamins and / or minerals.

[0175] Packaged cultured meat products are also provided, which contain or are derived from the cells or cell lines of the present invention.

[0176] Guide RNA and kits

[0177] In another aspect, the present invention provides guide RNAs that individually or in combination target the sequences of SEQ ID NO.5, or SEQ ID NO.8, or SEQ ID NO.9, or SEQ ID NO.10, or SEQ ID NO:28, or SEQ ID NO.29, or SEQ ID NO.30. In additional embodiments, the present invention provides guide RNAs according to any of the foregoing embodiments of the present invention for use in a method of producing modified cells according to any of the foregoing embodiments of the present invention. In related embodiments, the present invention provides guide RNAs according to the foregoing embodiments of the present invention, wherein the modified cells are modified cells according to any of the foregoing embodiments of the present invention.

[0178] In additional embodiments, the present invention provides a kit of parts that contains at least one of the guide RNAs described above. In one embodiment, the guide RNA may be a chemically synthesized sgRNA. In related embodiments, the chemically synthesized sgRNA can be used in combination with a recombinant purified Cas9 protein for CRISPR.

[0179] As described above, in some embodiments, the methods of the invention use gene editing, which is performed using a sequence-specific endonuclease that targets one or more genes in a target animal cell. Also as explained, Cas9 and gRNA can be included in a single or two expression vectors. The sgRNA targets one or more gene nucleic acid sequences.

[0180] Accordingly, in another aspect of the invention, there is provided a nucleic acid construct comprising a nucleic acid sequence encoding at least one DNA-binding domain that can bind to one or more genes. In one embodiment, the porcine NF2 gene comprises the sequence of SEQ ID NO.1 (wild boar) (Sus scrofa) or SEQ ID NO.3 (wild boar - Large white breed) (Sus-scrova-Largewhite breed) or a functional variant, homolog, or ortholog thereof, as described herein. In this embodiment, the porcine Merlin protein comprises the sequence of SEQ ID NO.2 (wild boar) or SEQ ID NO.4 (Sus-scrova-Largewhite variant) or a functional variant, homolog, or ortholog thereof, as described herein. In one embodiment, the bovine NF2 gene comprises the sequence of SEQ ID NO.6 (cattle) (Bos taurus) or a functional variant, homolog, or ortholog thereof, as described herein. In this embodiment, the bovine Merlin protein comprises the sequence of SEQ ID NO.7 (cattle) (Bos taurus) or a functional variant, homolog, or ortholog thereof, as described herein.

[0181] In one embodiment, the nucleic acid sequence encodes at least one protospacer element.

[0182] In one embodiment, the construct further comprises a nucleic acid sequence encoding a CRISPR RNA (crRNA) sequence, wherein the crRNA sequence comprises a protospacer element sequence and additional nucleotides. In one embodiment, the construct further comprises a nucleic acid sequence encoding a trans-activating RNA (tracrRNA).

[0183] In additional embodiments, the construct encodes at least one single-guide RNA (sgRNA), wherein the sgRNA comprises a tracrRNA sequence and a crRNA sequence, and wherein the sgRNA targets the sequence of SEQ ID NO.5 listed herein. The PAM sequence is also shown in the section entitled Sequence Listing. The sgRNA can be used to manipulate animal cells. In another aspect of the invention, there is provided a nucleic acid construct comprising a DNA donor nucleic acid, wherein the DNA donor nucleic acid is operably linked to a regulatory sequence. The regulatory sequence can be one or more of the following: an intron, a promoter, and / or a terminator.

[0184] Cas9 and sgRNA can be combined or in separate expression vectors (or nucleic acid constructs, these terms being used interchangeably). Similarly, Cas9, sgRNA, and donor DNA sequences can be combined or in separate expression vectors. In other words, in one embodiment, isolated animal cells are transfected with a single nucleic acid construct comprising sgRNA and Cas9 or sgRNA, Cas9, and donor DNA sequences, as described in detail above. In an alternative embodiment, isolated animal cells are transfected with two or three nucleic acid constructs, a first nucleic acid construct comprising at least one sgRNA as defined above, a second nucleic acid construct comprising Cas9 or a functional variant or homolog thereof, and optionally a third nucleic acid construct comprising a donor DNA sequence as defined above. The second and / or third nucleic acid constructs can be transfected before, after, or simultaneously with the first and / or second nucleic acid constructs. The advantage of a separate second construct comprising a Cas protein is that a nucleic acid construct encoding at least one sgRNA can be paired with any type of Cas protein, as described herein, and is thus not limited to a single Cas function (as is the case when Cas and sgRNA are both encoded on the same nucleic acid construct).

[0185] In one embodiment, the construct as described above is operably linked to a promoter, such as a constitutive promoter.

[0186] In another embodiment, the nucleic acid construct further comprises a nucleic acid sequence encoding a CRISPR enzyme. Preferably, the CRISPR enzyme is a Cas protein. More preferably, the Cas protein is Cas9 or a functional variant thereof.

[0187] In an alternative embodiment, the nucleic acid construct encodes a TAL effector. Preferably, the nucleic acid construct further comprises a sequence encoding an endonuclease or its DNA cleavage domain. More preferably, the endonuclease is FokI.

[0188] In another aspect of the invention, single-guide (sg) RNA molecules are provided, wherein the sgRNA comprises a crRNA sequence and a tracrRNA sequence. In one embodiment, the sgRNA molecule can comprise at least one chemical modification, such as enhancing its stability and / or binding affinity to the target sequence or the binding affinity between the crRNA sequence and the tracrRNA sequence. For example, the crRNA can comprise phosphorothioate backbone modifications such as "-fluoro" ("-F"), "-O-methyl" ("-O-Me"), and S-constrained ethyl (cET) substitutions.

[0189] In additional embodiments, the nucleic acid construct may further comprise at least one nucleic acid sequence encoding a ribonuclease cleavage site. Preferably, the ribonuclease is Csy4 (also known as Cas6f). When the nucleic acid construct comprises multiple sgRNA nucleic acid sequences, the construct may comprise the same number of ribonuclease cleavage sites. In another embodiment, the cleavage site is " of the sgRNA nucleic acid sequence". Thus, each sgRNA nucleic acid sequence is flanked by a ribonuclease cleavage site. The term "variant" refers to a nucleotide sequence in which the nucleotides are substantially the same as one of the above sequences. Variants can be achieved by modifying, for example, inserting, substituting, or deleting one or more nucleotides. In a preferred embodiment, the variant has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to any one of the above sequences. In one embodiment, the sequence identity is at least 90%. In another embodiment, the sequence identity is 100%. Sequence identity can be determined by any of the known sequence alignment programs in the art.

[0190] The present invention also relates to a nucleic acid construct comprising a nucleic acid sequence operably linked to a suitable animal promoter. Suitable animal promoters can be constitutive or strong promoters, or tissue-specific promoters. In one embodiment, suitable animal promoters are selected from, but not limited to, PGK, CMV, EF1a, CAG, SV40, and Ubc.

[0191] The nucleic acid construct of the present invention may further comprise a nucleic acid sequence encoding a CRISPR enzyme. In a particular embodiment, Cas9 is codon-optimized Cas9. In another embodiment, the CRISPR enzyme is a protein from a class 2 candidate protein family, such as C2c1, C2C2, and / or C2c3. In one embodiment, the Cas protein is from Streptococcus pyogenes. In alternative embodiments, the Cas protein can be from any one of Staphylococcus aureus, Neisseria meningitides, or Streptococcus thermophilus.

[0192] The term "functional variant" of Cas9 as used herein refers to a variant Cas9 gene sequence or a portion of a gene sequence that retains the biological function of the complete non-variant sequence, e.g., acting as a DNA endonuclease, or recognizing or / and binding to DNA. Functional variants also include variants of a target gene that have sequence alterations that do not affect function, such as non-conserved residues. Also included are variants that are substantially identical to the wild-type sequences shown herein, i.e., having only some sequence variations, e.g., in non-conserved residues, and having biological activity.

[0193] In additional embodiments, the Cas9 protein has been modified to enhance activity. For example, in one embodiment, the Cas9 protein can comprise a D10A amino acid substitution, and this nickase only cleaves the DNA strand that is complementary to and recognized by the gRNA. In an alternative embodiment, the Cas9 protein can alternatively or additionally comprise an H840A amino acid substitution, and this nickase only cleaves the DNA strand that does not interact with the sRNA. In this embodiment, Cas9 can be used with a pair (i.e., two) of sgRNA molecules (or constructs expressing such a pair of sgRNAs), and thus can cleave target regions on opposite DNA strands, potentially increasing specificity by 100 - 1500-fold. In additional embodiments, the Cas9 protein can comprise a D1135E substitution. The Cas9 protein can also be a VQR variant. Alternatively, the Cas protein can comprise mutations in both the HNH and RuvC-like nuclease domains and thus be catalytically inactive. Such a catalytically inactive Cas protein can be used to block the transcription elongation process rather than cleave the target strand, and when co-expressed with an sgRNA molecule, results in loss of function of the incompletely translated protein. An example of a catalytically inactive protein is dead Cas9 (dCas9) caused by point mutations in the RuvC and / or HNH nuclease domains.

[0194] In additional embodiments, the Cas protein (e.g., Cas9) can be further fused with a repressive effector (e.g., a histone modification / DNA methylase or a cytidine deaminase) to effect site-directed mutagenesis. In the latter, the cytidine deaminase does not induce dsDNA breaks but mediates the conversion of cytidine to uridine, thus effecting a C to T (or G to A) substitution.

[0195] In additional embodiments, the nucleic acid construct comprises a ribonuclease. Preferably, the ribonuclease is Csy4 (also known as Cas6f), more preferably codon-optimized csy4. In one embodiment, when the nucleic acid construct comprises a Cas protein, the nucleic acid construct can comprise a sequence for expressing the ribonuclease, e.g., Csy4 expressed as a C-terminal P2A fusion (serving as a self-cleaving peptide) to the Cas protein (e.g., Cas9).

[0196] In one embodiment, the Cas protein, ribonuclease, and / or ribonuclease-Cas fusion sequence can be operably linked to a suitable animal promoter. Suitable animal promoters have been described above, but in one embodiment, can be PGK, CMV, EF1a, CAG, SV40, and Ubc.

[0197] Suitable methods for producing CRISPR nucleic acids and vector systems are known, such as those disclosed in Ran et al., 2013, Nat Protoc 8, 2281–2308 (2013).

[0198] In a further aspect of the invention, there is provided an isolated animal cell transfected with at least one nucleic acid construct as described herein. In one embodiment, an isolated animal cell is transfected with at least one nucleic acid construct as described herein and a second nucleic acid construct, wherein the second nucleic acid construct comprises a nucleic acid sequence encoding a Cas protein, preferably Cas9 protein or a functional variant thereof. Preferably, the second nucleic acid construct is transfected before, after, or simultaneously with the first nucleic acid construct as described herein.

[0199] In an alternative aspect of the invention, the nucleic acid construct comprises at least one nucleic acid sequence encoding a TAL effector. Targeted nucleases, such as meganucleases, ZNFs, TALENs, CRISPR nucleases, and their derivatives, such as (but not limited to) Prime Editors, base editors, CRISPRi, etc., can form part of the present invention.

[0200] Preferably, the nucleic acid encoding the sgRNA and / or the nucleic acid encoding the Cas protein are integrated in a stable form.

[0201] The scope of the present invention also includes the use of the above nucleic acid constructs (CRISPR constructs) or sgRNA molecules in any of the above methods. For example, there is provided the use of the above CRISPR constructs or sgRNA molecules to modulate the activity of one or more genes as described herein. In particular, as described herein, the CRISPR constructs can be used to generate loss-of-function or over-activated alleles.

[0202] Unless otherwise defined herein, scientific and technical terms related to this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. While the foregoing disclosure provides a general description of the subject matter encompassed within the scope of this disclosure, including methods for preparing and using this disclosure and its best mode, the following examples are provided to further enable those skilled in the art to practice this disclosure. However, those skilled in the art will understand that the details of these examples should not be construed as limiting the invention, and the scope of the invention should be understood from the appended claims of this disclosure and their equivalents. Given this disclosure, various other aspects and embodiments of this disclosure will be apparent to those skilled in the art.

[0203] All documents mentioned in this specification are hereby incorporated by reference in their entirety, including references to gene accession numbers, scientific publications, and patent publications.

[0204] As used herein, "and / or" shall be regarded as a specific disclosure of each of the two designated features or components, whether with or without the other. For example, "A and / or B" shall be regarded as a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, as if each were listed separately herein. Unless otherwise indicated by the context, the descriptions and definitions of the above features are not limited to any specific aspect or embodiment of the invention and apply equally to all aspects and embodiments described.

[0205] The present invention is further illustrated in the following non-limiting examples.

[0206] Examples

[0207] Example 1: Determination of the Doubling Time of NF2 Gene-Knockout Cells

[0208] Figure 1 and Figure 2 shows the doubling time of NF2 gene-knockout cells under suspension conditions.

[0209] Methods

[0210] The doubling time was evaluated after transferring the control cell pool (transfected with only Cas9, no sgRNA) and the NF2 knockout cell pool from adherent growth conditions to suspension growth conditions. Data points were from conical flasks in triplicate and were measured over 4 growth passages.

[0211] Results

[0212] Shows that the average doubling time was reduced by 42% (only Cas control: 46 hours, NF2 knockout cell pool: 27 hours). Figure 1 Shows the differences between the control cell pool (transfected with only Cas9, no sgRNA) and the NF2 knockout cell pool for each of the 4 generations, Figure 2 Shows the collective grouping of data points for both control cells and N2 knockout cells.

[0213] Example 2: NF2 Gene Editing Efficiency

[0214] Methods

[0215] Evaluation of NF2 knockout efficiency in a pool of porcine myoblast cells transfected with Cas9 protein and sgRNA targeting the NF2 gene. After two time points post-transfection and a 4-generation suspension growth study in triplicate flasks, DNA was isolated from the cell pool, amplified by PCR of the NF2 gene, and then the PCR amplicons were subjected to Sanger sequencing, and the sequencing results were analyzed using the ICE analysis tool (ICE.synthego.com) to determine the knockout efficiency.

[0216] Results

[0217] As Figure 3 shown, the evaluation of NF2 knockout efficiency in the porcine myoblast cell pool indicated that high efficiency was retained over time. This indicates that NF2 gene knockout is stable over 4 generations.

[0218] Example 3: Functional knockout of the NF2 gene encoding the Merlin protein preserves the mesenchymal cell phenotype and enhances the adipogenic differentiation potential of immortalized porcine myoblast cell lines.

[0219] A major challenge in the cultivated meat industry is to develop a rapidly and continuously growing number of cell lines to produce large amounts of nutritious cell-based proteins for meat production. While simultaneously retaining the ability of skeletal muscle-derived progenitor cells to fuse into fibroblasts to provide higher-order structured meat or to produce intramuscular fat content associated with traditional meat products. This dynamic represents a critical interface between innate biological capabilities and commercial feasibility, which is one of the key blocks in the cultivated meat pipeline. In agriculturally relevant species, skeletal muscle tissue contains a heterogeneous mixture of cell types (muscle, mesenchymal stem cells (MSCs), nerves, immune cells, vasculature, etc.), which act in concert in response to environmental stimuli to adjust tissue content and maintain physiological homeostasis. Specifically in cultivated meat, protein content and fat accumulation are relevant. Maintaining this ability in cultivated meat cell lines requires maintaining this inherent heterogeneity when different nutritional and physiological requirements are imposed on the cells. Here, by introducing a functional knockout of the NF2 gene encoding the merlin protein and alleviating its inhibitory effect on cell growth and survival pathways, we successfully preserved a heterogeneous cell population, including MSCs that are crucial for fat production in muscle throughout the entire adaptation and growth process of suspension cell culture (a key process in cultivated meat cell line development).

[0220] MSCs in skeletal muscle tissue are diverse both in terms of individual heterogeneity and their developmental potential to undergo cellular and tissue lineage specialization. MSCs in muscle are typically identified by flow cytometry using the cell surface markers CD29 and CD90, and retaining this population enables muscle-derived cells to retain a plastic element in their phenotype. A subset of MSCs in skeletal muscle has been described as fibro-adipogenic progenitors (FAPs), which are responsible for intramuscular fat deposition. In flow cytometry, FAPs are also CD90+ and PDGFRa+, and are prone to adipogenic differentiation under in vivo nutritional and physiological cues or chemical induction in vitro. Here, we show that adapting our adherent immortalized muscle-derived cells to suspension culture can improve this mesenchymal CD90+ cell population ( Figure 4 a, d), and this change in the cell culture environment restricts the functional capacity of these cells to undergo adipogenic differentiation and accumulate intracellular fat ( Figure 4 h, i), thereby limiting the nutritional value potential of these cells. However, by introducing a functional knockout of the NF2 gene in the same immortalized cell line and adapting to the suspension protocol, we were able to retain this CD90+ population. Furthermore, compared to the unedited NF2(+ / +) parental, we were then able to demonstrate the retention of cell plasticity associated with muscle tissue in our NF2 knockout cells through enhanced lipid accumulation and adipogenic differentiation after in vitro chemical induction ( Figure 4 h, i). In the context of cultured meat, a functional NF2 gene deletion allows muscle-derived cells to grow rapidly in suspension culture while also maintaining the cell phenotype and plastic state required for intramuscular fat accumulation, as achieved in vivo. This will contribute to the development of cultured meat products with higher nutritional value and fat content without increasing the complexity of the manufacturing process, thus providing a nutritionally equivalent and environmentally friendly suitable alternative to traditional meat.

[0221] Example 4: Functional knockout of the NF2 gene encoding the Merlin protein can be achieved in multiple cell types with different genetic backgrounds in different species.

[0222] Figure 5 The editing efficiency of various cell types is shown, and it is shown that the editing is retained over time.

[0223] Method:

[0224] Cells were edited by nucleofection with synthetic single-guide RNA and Streptococcus pyogenes Cas9 protein. Note that the same guide RNA was used between breeds of a given species (e.g., Wagyu and Angus variants) and cell types (e.g., porcine myoblasts and porcine ADSCs).

[0225] Verify the editing efficiency in the cell pool at 2 - 3 edited time points to screen for enrichment or depletion of the desired mutation. Enrichment of the target mutation indicates a positive effect of the given mutation on cell growth, while depletion indicates an adverse effect on cell health or growth. Measure the editing efficiency by PCR amplification of the target region, Sanger sequencing, and ICE analysis of the Sanger sequencing files (Synthego).

[0226] In some cases, cells are pre - edited with sgRNAs targeting the P53, RB1, and / or HRAS genes (Seq ID No.11, 12, 13, 14, 15, 16, 17, 18, 19) before adding NF2 editing, or cells are edited simultaneously in these genes and in the NF2 gene to immortalize the cell line (see patent application PCT / GB2023 / 052528). For porcine and bovine HRAS, the G12V mutation is generated by adding ssODN homologous templates (Seq ID No.20, 21, 32, 33). In some cases, cells are immortalized by stable transfection with a lentiviral construct containing an over - active HRAS variant and simian virus 40 large T antigen prior to NF2 editing (cf. Hahn et al., 1999; https: / / doi.org / 10.1038 / 22780 and patent application PCT / GB2023 / 052528).

[0227] Please note that Figure 3 is equivalent to Figure 5 B

[0228] Results:

[0229] The NF2 gene can be effectively edited in different cell backgrounds, and this editing is retained in a mixed cell population. Cells are edited using a specified sgRNA targeting NF2, and the editing efficiency is measured at two to three time points after editing and after functional assays (where applicable). Unless otherwise stated, cells are edited in the adherent state. The cell types edited are (a) porcine primary myoblasts (two independent editing reactions); (b) porcine CRISPR-immortalized myoblasts (one editing reaction, generated from triplicate flasks during growth assays); (c) porcine virus-immortalized myoblasts; (d) suspension-growing porcine virus-immortalized ADSCs; (e) porcine CRISPR-immortalized ADSCs (data points from one editing reaction, two data points obtained from independent flasks during growth assays); (f) Angus bovine variant CRISPR-immortalized ADSCs. Note that in this experiment, cells were co-edited with P53 and RB1 sgRNAs to ensure simultaneous immortalization. Data points from one editing reaction, three data points taken from independent flasks during growth assays; (g) Angus bovine variant CRISPR-immortalized ADSCs. Note that in this experiment, cells were co-edited with P53, RB1, and HRAS sgRNAs (plus HRAS G12V repair template) to ensure simultaneous immortalization; (h) Angus bovine variant CRISPR-immortalized (P53 - / - , RB1 - / - ) myoblasts. Data points from one editing reaction, three data points taken from independent flasks during growth assays; (i) Angus bovine variant CRISPR-immortalized (P53 - / - , RB1 - / - , HRAS G12V / - ) myoblasts; (j) Wagyu bovine variant CRISPR-immortalized ADSCs. Note that in this experiment, cells were co-edited with P53 and RB1 sgRNAs to ensure simultaneous immortalization. Data points from two independent editing reactions, one data point from one replicate flask at the end of the growth assay. (k) Wagyu bovine variant CRISPR-immortalized myoblasts. Note that in this experiment, cells were co-edited with P53 and RB1 sgRNAs to ensure simultaneous immortalization. Data points from two independent editing reactions, one data point from one replicate flask at the end of the growth assay.

[0230] Example 5: NF2 gene knockout can provide a growth advantage in adhesion

[0231] Figure 6 Shows the doubling time assessment of NF2 knockout cell lines compared to control lines under adherent growth conditions.

[0232] Method:

[0233] Cells were grown adherently for multiple generations to obtain doubling time data and / or cumulative number of generations. Cells were seeded at 2000 - 4000 cells / cm 2 into flasks coated with Matrigel or uncoated (as shown). Cells were counted and passaged every 3 - 4 days. The culture medium for myoblasts was DMEM with 4.5 g / L glucose, 20% FBS, 2 mM L - glutamine, 5 ng / μL FGF2. The culture medium for ADSCs was DMEM, 1 g / L glucose, 10% FBS, 2 mM L - glutamine, 5 ng / μL FGF.

[0234] Results:

[0235] For the growth assay, cells were seeded into flasks in triplicate and grown for 5 - 8 generations on flasks coated with Matrigel or uncoated, as indicated. Doubling times were compared to one or two control lines (Ctrl). Unpaired two - tailed T - tests were used to determine the significance level between groups, where *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. (a) Doubling times of the Angus cattle variant myoblast pool between 8 generations. (b) Combined doubling time data for all 8 generations in (a). The average doubling time of Ctrl was 29.3 hours, while that of NF2 - / - cells was 27.1 hours. (c) Doubling times of the Angus cattle variant ADSC pool between 8 generations. (d) Combined doubling time data for all 8 generations in (c). The average doubling time of Ctrl was 25.2 hours, while that of NF2 - / - cells was 23.1 hours. (e) Doubling times of the Wagyu cattle variant myoblast pool between 5 generations. (f) Combined doubling time data for all 5 generations in (e). Unedited controls grown on plastic, P53 - / - / RB1 - / - and P53 - / - / RB1 - / - / NF2 - / - cells had average doubling times of 37, 24.9, and 21.4 hours, respectively. Unedited controls grown on matrigel, P53 - / - / RB1 - / - and P53 - / - / RB1 - / - / NF2 - / - cells had average doubling times of 34.6, 24.3, and 24.3 hours, respectively.

[0236] Example 6: NF2 gene knockout can provide a growth advantage in suspension adaptation

[0237] Figure 7 Shows the doubling time assessment of NF2 knockout cell lines compared to control lines under suspension growth conditions.

[0238] Methods:

[0239] For the suspension growth assay, cells were transferred from adherent flasks to conical flasks and grown at 100 rpm in suspension medium at an inoculation density of 100,000 cells / mL. Cells were counted and passaged every 3 - 4 days.

[0240] Results:

[0241] For suspension adaptation, cells were seeded in triplicate in conical flasks and grown for 4 - 5 passages in suspension medium as indicated. Doubling times were compared to the control line (Ctrl). Unpaired two - tailed T - tests were used to determine the significance levels between groups, where *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. (a) Doubling times of porcine myoblast cell pools between 4 passages. (b) Pooled doubling time data for all 4 passages in (a). The mean doubling time of Ctrl was 45.8 hours, while that of NF2 - / - cells was 27 hours. (c) Doubling times of porcine ADSC cell pools between 5 passages. (d) Pooled doubling time data for all 5 passages in (c). The mean doubling time of Ctrl was 118.2 hours, while that of NF2 - / - cells was 25.1 hours. (e) Doubling times of Angus cattle variant myoblast cell pools between 5 passages. (f) Pooled doubling time data for all 5 passages in (e). The mean doubling time of Ctrl was 72.2 hours, while that of NF2 - / - cells was 62.9 hours. (g) Doubling times of Angus cattle variant ADSC cell pools between 5 passages. (h) Pooled doubling time data for all 5 passages in (g). The mean doubling time of Ctrl was 36.7 hours, while that of NF2 - / - cells was 25.3 hours.

[0242] Example 7: NF2 gene knockout provides a stronger growth advantage in suspension than other Hippo pathway genes

[0243] Figure 8 The enrichment of cells with different gene mutations in the Hippo pathway was compared in a growth competition assay.

[0244] Methods:

[0245] Porcine CRISPR immortalized suspension cells (P53 - / - 、RB1 - / - 、HRASG12V / - ) In one of the three Hippo pathway genes (NF2 / LATS1 / LATS2), editing was performed in separate editing reactions using three sgRNAs for each gene (Seq ID No.22, 23, 24, 25, 26, 27, 28, 29, 30). After editing, the edited cell pools were combined with other mutant cell pools (data not shown) in a conical flask and grown in suspension at 100 rpm for ten days. Samples were collected on day 1 after combining the cell pools and on day 6 (the day of peak cell density) for DNA extraction and amplification of the target gene in a multiplex PCR reaction. The frameshift mutation frequency of each target gene in the cell pool was analyzed by next-generation sequencing. The percentage change in the frameshift mutation in the NGS reads between the two time points (as an approximation of functional gene knockout) was compared to screen for mutations that provide a stronger growth advantage.

[0246] Results:

[0247] When combined in one flask, NF2 knockout cells were enriched faster than cells with other mutations in the Hippo pathway, as seen by the higher increase in next-generation sequencing reads of frameshifts during the analysis period.

[0248] Table 1 Sequences

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

Claims

1. A cultured animal cell that has a genetic modification in the NF2 gene or a modification of the activity of the Merlin protein, wherein the animal is an animal species suitable for human or animal consumption.

2. The cultured animal cell according to claim 1, wherein the modification reduces the doubling time of the cell by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.

3. The cultured animal cell according to claim 1 or 2, wherein the animal is selected from pigs, cows, poultry, sheep, goats, equids, camelids, fish, crustaceans or molluscs.

4. The cultured animal cell according to any one of the preceding claims, wherein the animal cell is a somatic cell.

5. The cultured animal cell according to claim 4, wherein the animal cell is selected from one of the following cell types: myoblasts, fibroblasts, myofibroblasts, adipose-derived stem cells, epithelial cells, mesenchymal stem cells, satellite cells, iPSCs or hepatocytes.

6. The cultured animal cell according to any one of the preceding claims, wherein the expression of the NF2 gene or the activity of the Merlin protein is modified in the animal cell by one or more of the following methods: 1) Modification at the gene level by: a. Knockout or reduction of activity / transcription / translation levels by editing the coding sequence, promoter, intron, regulatory region; b. RNA-guided DNA methylation; or c. Transcriptional activation or repression using CRISPRa or CRISPRi or similar target-specific methods; d. Knockout or reduction of activity / transcription / translation levels by non-directed means, such as radiation or chemical mutagenesis; 2) Modification at the post-transcriptional level (post-transcriptional gene silencing) by: a. RNAi or siRNA to reduce the translation of mRNA into protein; or b. Site-specific nucleases to modify or cleave mRNA, such as CRISPR / Cas13a; 3) Modification at the post-translational level (protein destruction) by: a. Containing an activity-blocking / reducing molecule, wherein the activity-blocking / reducing molecule is a small molecule, antibody, etc.; or b. Containing a protein degradation component, wherein the protein degradation component is a specialized protease, exoprotease or endoprotease.

7. The cultured animal cell according to any one of claims 1 to 6, wherein the animal cell has a genetic modification in the NF2 gene.

8. The cultured animal cell according to claim 7, wherein the genetic modification in the NF2 gene is a loss-of-function modification or results in a reduction in function.

9. The cultured animal cell according to claim 8, wherein the loss-of-function modification includes knockout of the gene or loss of protein function.

10. The cultured animal cell according to any one of the preceding claims, wherein the modification is introduced by targeted genomic modification or random mutagenesis or by spontaneous mutation.

11. The cultured animal cells according to any one of the foregoing embodiments, wherein the modification is located in the promoter region or coding region of one or more genes.

12. The cultured animal cells according to any one of the foregoing embodiments, wherein the modification is introduced using targeted genomic modification, optionally introduced using a targeted endonuclease.

13. The cultured animal cells using an endonuclease according to claim 12, wherein the endonuclease is optionally selected from TALEN, ZFN or CRISPR, optionally CRISPR / Cas9.

14. The cultured animal cells according to any one of the foregoing claims, wherein the cultured animal cells further comprise at least one additional gene modification to manipulate genomic surveillance, cell cycle control and / or cell death control pathways.

15. The cultured animal cells according to claim 14, wherein the at least one additional gene modification is present in one or more of the following genes: RB1, TP53 and / or RAS genes.

16. The cultured animal cells according to claim 15, wherein the animal cells have a gene modification in RB1.

17. The cultured animal cells according to claim 15, wherein the animal cells have a gene modification in TP53.

18. The cultured animal cells according to claim 15, wherein the animal cells have a gene modification in the RAS gene.

19. The cultured animal cells according to claim 15, wherein the animal cells have gene modifications in RB1 and TP53.

20. The cultured animal cells according to claim 15, wherein the animal cells have gene modifications in RB1 and the RAS gene.

21. The cultured animal cells according to claim 15, wherein the animal cells have gene modifications in TP53 and the RAS gene.

22. The cultured animal cells according to claim 15, wherein the animal cells have gene modifications in RB1, TP53 and the RAS gene.

23. The cultured animal cells according to any one of claims 15 to 22, wherein the RAS gene is HRAS, NRAS or KRAS.

24. The cultured animal cells according to claim 23, wherein the RAS gene is HRAS.

25. A method for producing cultivated meat or cultured meat products, which comprises culturing the animal cells according to any one of claims 1 to 24.

26. A method for producing the cultured animal cells according to any one of claims 1 to 24.

27. The method according to claim 26, wherein the animal is selected from pigs, cows, poultry, sheep, goats, fish, equids, camelids, crustaceans or molluscs.

28. The method according to any one of claims 26 to 27, wherein the animal cells are somatic cells.

29. The animal cell according to claim 28, wherein the animal cell is selected from one of the following cell types: myoblasts, fibroblasts, myofibroblasts, adipose-derived stem cells, epithelial cells, mesenchymal stem cells, satellite cells, iPSCs or hepatocytes.

30. The method according to claims 26 to 29, wherein the modification is introduced using targeted genomic modification or random mutagenesis or by spontaneous mutation.

31. A cultivated or cultured animal tissue or a cultivated or cultured meat product, which comprises the modified cell according to any one of claims 1 to 24.

32. Use of the modified cultured animal cell according to any one of claims 1 to 24 for cell agriculture.

33. A method for producing an immortalized animal cell line, which comprises the method according to any one of claims 26 to 28, wherein the immortalized cell line comprises an NF2 gene modification.

34. A guide RNA, which targets the sequence of SEQ ID NO.5, or SEQ ID NO.8, or SEQ ID NO.9, or SEQ ID NO.10, or SEQ ID NO:28, or SEQ ID NO.29 or SEQ ID NO.30 alone or in combination.

35. A kit, which comprises at least one guide RNA according to claim 34.

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