Method of culturing an animal cell

Abstract

The invention relates to methods for culturing cells, and cultured animal cells for use in described method. Also provided is a modified animal cell and guide RNAs targeting genes to be modified in the modified and cultured cells of the invention.

Description

[0001] Method of Culturing an Animal Cell

[0002] Field of invention

[0003] The invention relates to a method of culturing an animal cell in cell culture which is serum, exogenous growth factor, and / or insulin free or wherein the cell culture medium is reduced in serum, at least one exogenous growth factors, and / or insulin and genetically manipulated cultured meat cells for use in the method.

[0004] Background

[0005] The world population is set to increase to almost 10 billion people within the next 50 years. As a result, there will be nearly two billion additional people to feed by 2050. This rising population will lead to an increase in global demand for meat by approximately 73% by the year 2050. The agricultural industry will have to scale, potentially doubling in size, to meet this demand. Of the earth’s habitable land, 39% is currently used to produce feed to rear livestock for the meat industry. It takes three years to rear a single cow for slaughter, or 6-12 months for pigs and poultry. Therefore, a large area of arable land is required to feed these animals to term. Currently, 80 billion animals are slaughtered each year for meat with 1 .2 billion slaughtered in the UK alone.

[0006] Cultivated meat has the potential to address the substantial global problems associated with livestock farming and the environmental impact of meat production along with animal welfare, food security and human health. Cultivated meat is a meat produced by in vitro cell cultures of animal cells. It is a form of cellular agriculture, with such agricultural methods being explored in the context of increased consumer demand for protein. Cellular agriculture relates to the production of animal-sourced foods from cell culture.

[0007] Cultivated meat is produced using tissue engineering techniques traditionally used in regenerative medicines and requires cell lines, usually stem cells. Stem cells are undifferentiated cells which have the potential to become many or all of the required kinds of specialized cell types. While pluripotent stem cells are often thought of as the ideal starting cell, the most prominent example of this subcategory of stem cell are embryonic stem cells which due to ethical issues are controversial for use in research. As a result, induced pluripotent stem cells (iPSCs) have been developed. iPSCs are multipotent blood and skin cells that are artificially regressed to a pluripotent state enabling them to differentiate into a greater range of cells. The alternative to iPSCs involves the use of multipotent adult stem cells which give rise to muscle cell lineages or unipotent progenitors which can differentiate into muscle cells. Favourable characteristics of stem cells which make them suitable for cultivated meat production include immortality, increased proliferative ability, lack of reliance on adherence, serum independence and easy differentiation into tissue. Stem cells used to generate cell lines can be collected from a primary source, i.e., through a biopsy on an animal under local anaesthesia and can also be established from secondary sources such as cryopreserved cultures. However, somatic cells isolated from tissues / organs often used in food consumption (e.g. muscle, fat, and fibroblasts) from agriculturally relevant species (e.g. pigs, cows, chickens) have a limited lifespan when grown in vitro. Although it is possible to isolate primary cell lines from pigs (myoblasts, myofibroblasts, fibroblasts, adipose derived stem cells and epithelial cells) the ability to propagate these cell lines with efficient doubling times and for long term is not feasible.

[0008] In vitro culturing of cells is typically enabled by growing cells in specific growth media which supplies them with vital nutrients, such as amino acids, lipids, carbohydrates but also signalling molecules such as hormones and growth factors to trigger proliferation and support cell growth and function. Traditionally, many cell culture media also contain serum, such as fetal bovine serum (FBS), which is used as a source for growth factors, hormones, lipids, and minerals, amongst others for the cells. However, these traditional media formulations used in biomedical research and industry are not suitable for the cultivated meat sector for several reasons. Growth factors and serum are expensive ingredients and are not economical for production of a food product. Components like serum are animal derived and usually involve slaughtering of said animals, which raises ethical concerns around the cultivated meat products. Serum is a naturally derived product which leads to batch-to-batch variations but also supply chain issues, which makes it unsuitable for industrial process development. For the cultivated meat industry, it is therefore vital to reduce the need for expensive, animal derived components like growth factors and serum from media formulations without affecting the growths of cells.

[0009] There is a need to reduce cell doubling times (also in the context of suspension culture) and improve the economic viability of cultured meat by removing growth factors and serum from cell culture media formulations. The invention addresses this need by providing manipulated animal cells that comprise modification of endogenous genes or expression of exogeneous nucleic acid constructs to overcome the cells’ need for growth factors and / or serum.

[0010] Summary of the invention

[0011] According to a first aspect of the invention, there is provided a method of culturing an animal cell comprising culturing the animal cell in a cell culture medium, wherein the cell culture medium is serum, exogenous growth factor, and / or insulin free or wherein the cell culture medium is reduced in serum, at least one exogenous growth factors, and / or insulin, and wherein the animal cell comprises a genetic modification in one or more of a RAS gene, the TP53 gene, the RB1 gene, the NF2 gene, the PTEN gene, the SPRY gene, the BAX gene and / or the BAK1 gene.

[0012] In one embodiment, the media comprises 2.5% serum or less. In one embodiment, the media comprises at least one exogenous growth factors at a concentration of 20 ng / mL or less.

[0013] In one embodiment, the cell culture medium comprises 100 pg / mL insulin or less.

[0014] In one embodiment, the animal is of an animal species suitable for human or animal consumption.

[0015] In one embodiment, the animal is selected from a pig, bovine, poultry, sheep, goat, fish, Camelidae, Equidae, crustaceans or mollusc.

[0016] In one embodiment, the animal cell is a somatic cell.

[0017] In one embodiment, the animal cell is selected from one of the following cell types: myoblast, fibroblast, myofibroblast, adipose derived stem cell, epithelial cell, mesenchymal stem cell, satellite cell, iPSC, or hepatocyte.

[0018] In one embodiment, the genetic modification in the animal cell is by any one or more of:

[0019] 1) gene level modification by: a. knock-out or reduced activity / transcription / translation levels via editing in coding sequences, promoters, introns, regulatory regions; b. RNA-directed DNA methylation; or c. transcription activation or repression using CRISPRa or CRISPRi or similar target specific methods; d. knock-out or reduced activity / transcription / translation levels via undirected means, for example radiation or chemical mutagenesis; e. overexpression of an endogenous nucleotide sequence

[0020] 2) post-transcription level (post-transcriptional gene silencing) modification by: a. RNAi or siRNA to reduce translation of mRNA into protein; or b. site specific nucleases to modify or cleave mRNA, such as CRISPR / Cas13a;

[0021] 3) post-translational level (protein disruption or activation) modification by: a. inclusion of activity blocking / reducing or enhancing molecules, wherein the activity blocking / reducing or enhancing molecules are small molecules, antibodies, or the like; or b. inclusion of protein degrading ingredients, wherein the protein degrading ingredients are specialised proteases, exoproteases, or endoproteases. c. Enhancing or reducing of protein activity through integration of activating or reducing functional mutations in the corresponding gene sequences. In one embodiment, the modification decreases 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%, or 95% or about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold or 100-fold in comparison to the unmodified animal cell.

[0022] In one embodiment, the modification increases cell size by at least about 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150% or 200% or about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold or 100-fold in comparison to the unmodified animal cell.

[0023] In one embodiment, the genetic modification is a loss of function modification or leads to reduction in function, wherein the loss of function modification comprises a knock-out of the gene or loss of protein function.

[0024] In one embodiment, the modification is introduced using targeted genome modification or randomised mutagenesis or by spontaneous mutation.

[0025] In one embodiment, the modification is in the promoter region or coding region of one or more genes.

[0026] In one embodiment, the modification is introduced using targeted genome modification, optionally using a targeted endonuclease.

[0027] In one embodiment, an endonuclease, wherein the endonuclease is selected from TALEN, ZFN or CRISPR, optionally CRISPR / Cas9.

[0028] In one embodiment, the animal cell comprises a genetic modification in a RAS gene, the PTEN gene and / or a SPRY gene.

[0029] In one embodiment, the SPRY gene is selected from SPRY1 , SPRY2, SPRY3, and / or SPRY4.

[0030] In one embodiment, the SPRY gene is SPRY2.

[0031] In one embodiment, the genetic modification in the TP53 gene, the RB1 gene, the NF2 gene, the PTEN gene and / or a SPRY gene is a loss of function modification.

[0032] In one embodiment, the loss of function modification comprises a knock-out of the gene.

[0033] In one embodiment, the genetic modification in the TP53 gene, the RB1 gene, the NF2 gene, the PTEN gene and / or a SPRY gene is a knock out genetic modification in one or both alleles. In one embodiment, the animal cell also has a genetic modification in a RAS gene.

[0034] In one embodiment, the RAS gene is HRAS, NRAS, or KRAS.

[0035] In one embodiment, the RAS gene is HRAS.

[0036] In one embodiment, the genetic modification to the RAS gene is an overexpression and / or hyperactivation of the gene or gene product.

[0037] In one embodiment, the method comprises continuous or batch culture of the modified animal cell.

[0038] In one embodiment, the method comprises the step of forming the animal cells into a tissue like structure.

[0039] In one embodiment, the animal cells are formed into a muscle tissue like structure.

[0040] In one embodiment, the modified animal cell is used in the production of cultivated meat or a cultured meat product.

[0041] According to a second aspect of the invention, there is provided a cultured animal cell for use in a method according to the invention.

[0042] In one embodiment, the cultured animal cell comprises a genetic modification in the PTEN gene and / or SPRY gene and wherein the animal is of an animal species suitable for human or animal consumption. It is described here for the first time that a functional knock-out of the PTEN gene and / or SPRY gene decreases doubling times in porcine and bovine myoblasts and Adipose-derived stem cells. It is therefore advantageously demonstrated that inactivation of the endogenous PTEN gene and / or SPRY gene is sufficient to speed up proliferation of porcine and bovine cell cultures. The commercial viability of cultured meat is greatly improved by the aforementioned advantages of increased proliferation, i.e. decreased doubling time. This may be measured against a control cell line which does not comprise the genetic modification in the PTEN gene and / or a SPRY gene, as appropriate.

[0043] In one embodiment, the SPRY gene is selected from a group consisting of SPRY1, SPRY2, SPRY3, and / or SPRY4.

[0044] In one embodiment, the SPRY gene is SPRY2. In one embodiment, the animal is selected from a pig, bovine, poultry, sheep, goat, Equidae, Camelidae, fish, crustaceans or mollusc.

[0045] In one embodiment, the animal cell is a somatic cell.

[0046] In one embodiment, the animal cell is selected from one of the following cell types: myoblast, fibroblast, myofibroblast, adipose derived stem cell, epithelial cell, mesenchymal stem cell, satellite cell, iPSC, or hepatocyte.

[0047] In one embodiment, the genetic modification in the PTEN gene and / or SPRY gene is a loss of function modification or leads to reduction in function.

[0048] In one embodiment, the loss of function modification comprises a knock-out of the PTEN gene and / or SPRY gene or loss of protein function.

[0049] In one embodiment, the modification is introduced using targeted genome modification or randomised mutagenesis or by spontaneous mutation.

[0050] In one embodiment, the modification is in the promoter region or coding region of one or more genes.

[0051] In one embodiment, the modification is introduced using targeted genome modification, optionally using a targeted endonuclease.

[0052] In one embodiment, the endonuclease is selected from meganucleases, TALEN, ZFN or CRISPR, optionally CRISPR / Cas9.

[0053] In one embodiment, the at least one additional genetic modification is in one or more of the following genes: RB1, TP53, NF2, and / or a RAS gene.

[0054] In one embodiment, the RAS gene is HRAS, NRAS, or KRAS.

[0055] According to a third aspect, there is provided a method of producing cultivated meat or a cultured meat product comprising culturing the animal cell according to an aspect of the invention.

[0056] According to a fourth aspect of the invention, there is provided a method of producing the cultured animal cell wherein the modification decreases the doubling time and / or increases cell size 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%, or 95% in comparison to the unmodified animal cell or about 2-fold, 3-fold, 4-fold, 5- fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90- fold or 100-fold in comparison to the unmodified animal cell.

[0057] According to a fifth aspect of the invention, there is provided cultivated or cultured animal tissue or a cultivated or cultured meat product comprising a modified cell according to an aspect of the invention.

[0058] According to a sixth aspect of the invention, there is provided a use of the modified cultured animal cell according to an aspect of the invention for cellular agriculture.

[0059] According to a seventh aspect of the invention, there is provided a guide RNA targeting the sequence for the PTEN gene or the sequence for one or multiple genes of the SPRY gene family in a cultured animal cell according to an aspect of the invention.

[0060] In one embodiment, the guide RNA targets the sequence for the PTEN gene and comprises a sequence according to SEQ ID NO: 19 or SEQ ID NO: 20.

[0061] In one embodiment, the guide RNA targets the sequence for the SPRY1 gene and comprises a sequence according to SEQ ID NO: 21 .

[0062] In one embodiment, the guide RNA targets the sequence for the SPRY2 gene and comprises a sequence according to SEQ ID NO: 22 or SEQ ID NO: 23.

[0063] In one embodiment, the guide RNA targets the sequence for the SPRY3 gene and comprises a sequence according to SEQ ID NO: 24.

[0064] In one embodiment, the guide RNA targets the sequence for the SPRY4 gene and comprises a sequence according to SEQ ID NO: 25.

[0065] According to an eighth aspect of the invention, there is provided a kit comprising at least one the guide RNA of an aspect of the invention.

[0066] According to a ninth aspect of the invention, there is provided a modified animal cell comprising a genetic modification in the BAK1 gene and / or the BAX gene, and wherein the animal is of an animal species suitable for human or animal consumption.

[0067] In one embodiment, the animal is selected from a pig, bovine, poultry, sheep, goat, Equidae, Camelidae, fish, crustaceans or mollusc.

[0068] In one embodiment, the animal cell is a somatic cell. In one embodiment, the animal cell is selected from one of the following cell types: myoblast, fibroblast, myofibroblast, adipose derived stem cell, epithelial cell, mesenchymal stem cell, satellite cell, iPSC, or hepatocyte.

[0069] In one embodiment, the genetic modification is a gene is a loss of function modification or leads to reduction in function.

[0070] In one embodiment, the genetic modification comprises a knock-out of the BaAK1 gene and / or the BaAX gene or loss of protein function.

[0071] In one embodiment, the modification is introduced using targeted genome modification or randomised mutagenesis or by spontaneous mutation.

[0072] In one embodiment, the modification is in the promoter region or coding region of one or more genes.

[0073] In one embodiment, the modification is introduced using targeted genome modification, optionally using a targeted endonuclease.

[0074] In one embodiment, the endonuclease is selected from meganuclease, TALEN, ZFN or CRISPR, optionally CRISPR / Cas9.

[0075] According to a tenth aspect of the invention, there is provided a method of producing cultivated meat or a cultured meat product comprising culturing the modified animal cell according to the invention.

[0076] According to an eleventh aspect of the invention, there is provided a method of conferring apoptosis resistance, improving cell viability, increasing cell density, increasing cell size and / or increasing cell lifespan of a cultivated or cultured animal cell suitable for human or animal consumption comprising cultivating or culturing animal cells comprising a genetic modification in the BAK1 gene and / or the BAX gene.

[0077] In one embodiment, the modification reduces apoptosis, increases viability, increases density, increasing cell size and / or increases lifespan 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%, or 95% in comparison to the unmodified animal cell, or about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold or 100-fold in comparison to the unmodified animal cell.

[0078] According to an twelfth aspect of the invention, there is provided a use of the modified cultured animal cell according to the invention for cellular agriculture. According to a thirteenth aspect of the invention, there is provided a guide RNA targeting the sequence for the BAK1 gene or the sequence for the BAX' gene in a cultured animal cell according to the invention.

[0079] In one embodiment, the guide RNA targets the sequence for the BAK1 gene and comprises a sequence according to SEQ ID NO: 42.

[0080] In one embodiment, the guide RNA targets the sequence for the BAX gene and comprises a sequence according to SEQ ID NO: 41 .

[0081] According to a fourteenth aspect of the invention, there is provided a kit comprising at least one guide RNA of the invention.

[0082] Figures

[0083] The invention is further described in the following non-limiting figures.

[0084] Figure 1 illustrates enrichment of PTEN and SPRY2 knockout mutants in a pooled cell competition assay in serum free suspension cell culture media lacking growth factors: (a) shows a schematic overview of the pooled cell competition assay. Cell lines mutated in relevant growth factor pathway related genes are pooled and grown in different media conditions over several passages. Enrichment or depletion of specific mutated cell lines in the pool over time can be determined using a multiplex PCR approach followed by next generation amplicon sequencing, (b-d) enrichment and depletion data of specific mutants in pooled experiment from duplicate Erlenmeyer flasks (n1 / n2) cultured in serum free suspension media containing no growth factors (b), serum free suspension media without IGF1 (c) and serum free suspension media without FGF2 (d). The fold change is shown on a Iog10 scale, where enrichment is represented by a darker shade of grey and white represents a depletion in functional gene knockout or knock-in within the cell pool relative to dO. Note that condition (b) was ended after passage 2 due to lack of cell growth, and flask n2 in condition (c) was grown for 7 passages only. PTEN mutants show strong enrichment in all conditions, SPRY2 mutants show enrichment under certain conditions.

[0085] Figure 2 illustrates the PTEN and SPRY2 genes can be edited efficiently in different cell types and species and the edits are retained in mixed cell populations cultured in media containing reduced growth factors: editing data from (a) edited porcine myoblast cell pools from different timepoints for (i) PTEN knockout cell pools (ii) SPRY2 knockout cell pools (iii) PTEN / SPRY2 double knockout cell pools; (b) Bovine var. Angus ADSCs from different timepoints for (i) PTEN KO (ii) SPRY2 KO (iii) PTEN / SPRY2 KO and PTEN / SPRY1-4 KO (iv) cell pools; (c) Bovine var. Wagyu myoblasts from different timepoints with a PTEN / SPRY2 KO. Figure 3 illustrates the PTEN and SPRY2 knockouts provide a growth advantage in suspension culture in porcine myoblast cells: (a) Doubling times (i) and cell densities (ii) of porcine myoblast cell pools over 11 passages in suspension media supplemented with 2.5% FBS, 5.25 ng / mL FGF, 8.75 ng / mL IGF1 , 1 ng / mL PDGF0 and 0.035 ng / mL HGF. (b) Doubling times (i) and cell densities (ii) of porcine myoblast cell pools over 10 passages in suspension media supplemented with 2.5% FBS, 1 .05 ng / mL FGF, 8.75 ng / mL IGF1 , 1 ng / mL PDGF0 and 0.035 ng / mL HGF. (c) Doubling times (i) and cell densities (ii) of porcine myoblast cell pools over 5 passages in suspension media supplemented with 2.5% FBS, 1 .05 ng / mL FGF, 1 .75 ng / mL IGF1 , 0.2 ng / mL PDGFp and 0.007 ng / mL HGF. (d) Combined doubling time data from (a). Average doubling time for the Ctrl (PTEN(+ / +) / SPRY2(+ / +)) is 26.9 h; 24.7 h PTEN cells; 24.2 cells; 24.5 h PTEN < / ) / SPRY2 cells, (e) Combined doubling time data from (b). Average doubling time for the Ctrl (PTEN <+ / +SPRY2 <+ / +>) is 34.1 h; 26.3 h PTEN ( cells; 29 h SPRY2 cells; 26.5 h PTEN <- / ) / SPRY2( / )cells, (f) Combined doubling time data from (c). Average doubling time for the Ctrl (PTEN <+ / +SPRY2 <+ / +>) is 34 h; 26.1 h PTEN cells; 28 h SPRY2 cells; 24.5 h PTEN( / ) / SPRY2( / )KO cells, (g) Doubling times (i) and cell densities (ii) of porcine myoblast cell pools over 5 passages in suspension media with 2.5% FBS and no additional growth factors followed by 5 passages in suspension media supplemented with 2.5% FBS and 1.05 ng / mL FGF2, as depicted by the dashed vertical line. Due to insufficient cell number, the Ctrl cells were not seeded beyond passage 3. Note that negative doubling times are not plotted, (h) Combined doubling time data from cells growing in suspension media supplemented with 2.5% FBS and 1 .05 ng / mL FGF2 in (g). Average doubling time for the PTEN cells is 33.4 h and 29.9 h PTEN cells.

[0086] Figure 4 illustrates the PTEN and SPRY1 / 2 / 3 / 4 knockouts provide a growth advantage and cell size increase in suspension media without growth factors in bovine var. Angus ADSC: (a) Doubling times (i) and cell densities (ii) of bovine ADSC Ctrl and PTENf7) pools over 10 passages in suspension media containing 2.5% FBS and 1 .05 ng / mL FGF2. (b) Doubling times (i) and cell densities (ii) of bovine ADSC Ctrl, PTEN^), SPRY2< / ) and PTEN(- / -) / SPRY1 (- / -) / SPRY2(- / -) / SPRY3(- / -) / SPRY4<- / -) pools over 7 passages in suspension media containing 2.5% FBS and no growth factors, (c) Doubling times (i) and cell densities (ii) of bovine ADSC Ctrl, PTEN( / ), SPRY2< / ) and PTEN<- / -) / SPRY1 (- / -) / SPRY2<- / ) / SPRY3( / ) / SPRY4( / )pools over 4 passages in serum free suspension media without the addition of growth factors, (d) Combined doubling time data from (a). Average doubling time for the Ctrl (PTEN (+ / +) / SPRY1-4(+ / +)) is 27.2 h and 26.2 h for PTEN( / )cells, (e) Combined doubling time data from (b). Average doubling time for the Ctrl (PTEN <+ / +VSPRY1-4 <+ / +>) is 75.5 h; 37.5 h PTEN cells; 37.7 h PTEN <- / - SPRY2 w cells; 35 h PTEN cells, (f) Combined cell size of bovine ADSC cell pools from the culture conditions in (a). Average cell size for the Ctrl (PTEN <+ / +SPRY1-4 <+ / +>) is 14.3 pm and 15 pm for PTEN( / )cells (g) Combined cell size of bovine ADSC cell pools from the culture conditions in (b). Average cell size for the Ctrl (PTEN(+ / +) / SPRY1-4(+ / +)) is 14.6 pm; 15.3 pm PTEN( / )cells; 15.3 pm PTEN <- / ) / SPRY2 cells; 15.2 pm PTEN w / SPRYI -4 cells, (h) Combined cell size of bovine ADSC cell pools from the culture conditions in (c). Average cell size for the Ctrl (PTEN(+ / +) / SPRY1-4(+ / +)) is 14 pm; 15.2 pm PTEN cells; 14.8 pm PTEN < / ) / SPRY2 w cells; 15 pm PTEN w / SPRYI -4 cells.

[0087] Figure 5 illustrates bovine var. Angus ADSC containing a PTEN / SPRY2 double knockout reach a higher maximum viable cell density in a suspension culture overgrowth study: the PTEN ' VSPRY2( / )cell line reaches higher viable cell densities than the PTEN(+ / +) / SPRY2(+ / +while maintaining similar viability.

[0088] Figure 6 illustrates bovine var. Wagyu myoblast cells containing a PTEN / SPRY2 double knockout have a lower doubling time in suspension culture conditions, a larger cell diameter, and grow well in low growth factor conditions: (a) Growth characteristics of Bovine var. Wagyu myoblast cells containing a PTEN / SPRY2 double knockout grown in suspension media with 2.5% FBS, 5 ng / mL FGF, 1 ng / mL PDGF0, 10 ng / mL IGF1 and 0.1 ng / mL HGF. Average doubling time Ctrl line: 41.8 h, average doubling time PTEN( / ) / SPRY2( / ): 33.3 h, average diameter Ctrl line: 12.9 pm, average diameter PTEN(_ / ) / SPRY2( / ): 14.2 pm. (b) Growth characteristics of Bovine var. Wagyu myoblast cells containing a PTEN / SPRY2 double knockout grown in suspension media with 2.5% FBS and 1 ng / mL FGF only. Average doubling time Ctrl line: 67.5 h, average doubling time PTEN( / ) / SPRY2( / ): 38.2 h, average diameter Ctrl line: 13.1 pm, average diameter PTEN( / ) / SPRY2( / ): 14.1 pm.

[0089] Figure 7 illustrates bovine var. Wagyu myoblast cells containing a PTEN / SPRY2 double knockout reach a higher maximum viable cell density in suspension culture: (a) Growth characteristics of Bovine var. Wagyu myoblast cells grown in suspension media with 2.5% FBS, 5 ng / mL FGF, 1 ng / mL PDGFp, 10 ng / mL IGF1 and 0.1 ng / mL HGF. (b) Growth characteristics of Bovine var. Wagyu myoblast cells grown in suspension media with 2.5% FBS and 1 ng / mL FGF only. Note that the PTEN( / ) / SPRY2 <-z-) cells reach higher peak density and have a larger diameter while maintaining viability.

[0090] Figure 8 illustrates porcine cells overexpressing an activated RAS protein have a growth advantage in growth factor free media: (a) Schematic design of RAS overexpression constructs used in this study, (b) Experimental design for RAS competition experiment: To determine which RAS isoform promotes cell proliferation the most, porcine myoblast cells overexpressing a RAS G12D isoform coupled to a fluorescent marker (eGFP-HRAS G12D, mCHERRY-NRAS G12D, mCERULEAN-KRAS G12D, indicated in figure by triangle, oval and rectangle respectively) were pooled and grown in triplicates in suspension media with or without growth factors and / or FBS for at least 5 passages. Percentage of cells expressing a given fluorophore in the pooled population were analysed each passage via flow cytometry. In each media condition, a control flask was taken along containing untransduced cells without an activated RAS overexpression to benchmark growth profile, (c) Doubling times (i), cell densities (ii), and % of cells expressing a given fluorophore in cell pool (iii) grown over 6 passages in suspension media containing 2.5% FBS and growth factor mix. (d) Doubling times (i), cell densities (ii), and % of cells expressing a given fluorophore in cell pool (iii) grown over 6 passages in suspension media containing 2.5% FBS but no growth factor mix. (e) Doubling times (i), cell densities

[0091] (ii), and % of cells expressing a given fluorophore in cell pool (iii) grown over 6 passages in suspension media containing neither growth factor mix nor FBS. Note that control flasks in (d)(i) and (e)(i) did not show cell proliferation in the given media condition, therefore no doubling times were plotted. Note that control flasks were ended after two passages in those conditions as there were not sufficient cells for reseeding as indicated in (d)(ii) and (e)(ii).

[0092] Figure 9 illustrates porcine cells overexpressing an activated HRAS protein have a growth advantage in growth factor free media: (a) Doubling times (i), cell densities (ii), and % of cells expressing eGFP in cell pool (iii) grown for 7 passages in suspension media containing 2.5% FBS and growth factor mix. (b) Doubling times (i), cell densities (ii), and % of cells expressing eGFP in cell pool

[0093] (iii) grown for 7 passages in suspension media containing 2.5% FBS but no additional growth factors, (c) Doubling times (i), cell densities (ii), and % of cells expressing eGFP in cell pool (iii) grown for 7 passages in suspension media containing no FBS or growth factors. Note that negative doubling times are not plotted, as e.g. for control line in (b)(i) and (c)(i), Also note that control cells died in media without growth factors as seen in (b)(ii) and (c)(ii). Note that the % of GFP positive cell population is increasing strongly in growth factor free conditions over the course of the experiment.

[0094] Figure 10 illustrates porcine cells overexpressing an activated KRAS protein have a growth: (a) Doubling times (i), cell densities (ii) and % of cells expressing mCerulean (iii) grown for 7 passages in suspension media containing 2.5% FBS and growth factor mix. (b) Doubling times (i), cell densities (ii) and % of cells expressing mCerulean (iii) grown for 7 passages in suspension media containing 2.5% FBS of cells and no additional growth factors. Note that negative doubling times are not plotted, as e.g. for control line in (b)(i), also note that control cells died in media without growth factors as seen in (b)(ii). (c) Average cell diameter of untransduced control cells compared to KRAS G12D cells (13.8 pm) compared to control cells (13.1 pm) across all media conditions and passages.

[0095] Figure 11 illustrates porcine cells overexpressing an activated HRAS protein can grow in bioreactors in growth factor and serum free media: (a) Overgrowth data from cells grown in Erlenmeyer flasks: Cell densities (i), glutamine and ammonia concentrations in spent media (ii), and glucose and lactate concentrations in spent media (iii). (b) Overgrowth data from cells grown in DASbox® Mini Bioreactor System. Cell densities (i), glucose and lactate concentrations in media (ii), glucose and lactate cell usage rates (iii), glutamine and ammonia concentrations in media (iv), glutamine and ammonia cell usage rates (v).

[0096] Figure 12 illustrates the growth advantage of cells overexpressing RAS G12D in suspension media without growth factors seen in porcine is consistent in bovine var. Angus cells: (a) Doubling times (i), cell densities (ii) and % of cells expressing each fluorophore (iii) grown in suspension media containing 2.5% FBS and additional growth factor mix. (b) Doubling times (i), cell densities (ii), % of cells expressing each fluorophore (Hi) and average doubling times across all 5 passages (iv) grown in suspension media containing 2.5% FBS but no additional growth factors. Average doubling time for Ctrl: 88.1 h, for HRAS G12D: 26.4 h, for KRAS G12D: 31.2 h, for NRAS G12D: 32.1 h. (c) Doubling times (i), cell densities (ii), % of cells expressing each fluorophore (iii) and average doubling times across all 5 passages (iv) grown in suspension media without FBS or additional growth factors. Average doubling time for HRAS G12D: 29.3 h, for KRAS G12D: 37.2 h, for NRAS G12D: 39.4 h. Note that negative doubling times are not plotted, as e.g. for control line in (c)(i), also note that control cells died in media without growth factors as seen in (c)(ii).

[0097] Figure 13 illustrates bovine var. Angus cells overexpressing an activated HRAS protein show comparable growth in media without growth factors as control cells in media with growth factors in overgrowth studies: (a) Cell densities (i), glutamine and ammonia levels in the media (ii), glucose and lactate levels in the media (iii), glutamine and ammonia cell usage rates (iv) and glucose and lactate cell usage rates (v) over 7 days growth period.

[0098] Figure 14 illustrates bovine var. Wagyu cells overexpressing an activated RAS protein have a growth advantage in growth factor free media: (a) Doubling times (i), cell densities (ii), % of cells expressing a given fluorophore in the pool (iii) and % of cells expressing eGFP in cells overexpressing HRAS G12D grown separately (iv) grown for 5 passages in suspension media containing 2.5% FBS and growth factor mix. (b) Doubling times (i), cell densities (ii), % of cells expressing a given fluorophore in the pool (iii) and % of cells expressing eGFP in cells overexpressing HRAS G12D grown separately (iv) grown for 5 passages in suspension media containing 2.5% FBS but no additional growth factors, (c) Doubling times (i), cell densities (ii), % of cells expressing a given fluorophore in the pool (iii) and % of cells expressing eGFP in cells overexpressing HRAS G12D grown separately (iv) grown for 5 passages in suspension media containing no FBS or growth factors. Note that negative doubling times are not plotted, as e.g. for control line in (c)(i), Also note that control cells died in media without FBS and growth factors as seen in (c)(ii). (d) Average doubling times across the passages of the assay comparing the control cells, the pooled cells and the eGFP-HRAS G12D cells alone in suspension media containing 2.5% FBS and additional growth factor mix (i), with Ctrl: 33.5 h, HRAS G12D: 30.9 h, Pool: 29.2 h; in suspension media with 2.5% FBS but no additional growth factors (ii), with Ctrl: 152.3 h, HRAS G12D: 31 h, Pool: 29.9 h; and in suspension media without FBS or additional growth factors (iii), with HRAS G12D: 30.1 h, Pool: 32.6 h;. Note that data from passage 3 was excluded from doubling time data analysis (e) Average cell diameter of untransduced control cells compared to HRAS G12D cells compared across all media conditions and passages, with Ctrl: 14.5 pm, HRAS G12D: 16.2 pm.

[0099] Figure 15 illustrates bovine var. Wagyu cells overexpressing an activated HRAS protein show growth in media without growth factors as control cells in media with growth factors in overgrowth studies: (a) Cell densities (i), glutamine and ammonia levels in spent media (ii), glucose and lactate levels in the media (iii), glutamine and ammonia cell usage rates (iv) and glucose and lactate cell usage rates (v) over 7 days growth period.

[0100] Figure 16 illustrates the PTEN / SPRY2 double knockout can be combined with an HRAS G12D overexpression in bovine var. Angus ADSC and leading to an additional cell size increase: (a) (b) (i) doubling times (ii) cell densities, (iii) pooled doubling times over all 4 passages and (iv) pooled cell size of bovine var. Wagyu adipose derived stem cell (ADSC) pools in suspension media containing 2.5% FBS and no growth factor. Average doubling time for the Ctrl is 41 .5 h and 42 h for the PTEN( / ) / SPRY2 <-7) cells. Average cell size for the Ctrl (PTEN <+7+> / SPRY2 <+ / +>) is 16.1 pm and 16.5 pm for PTEN < / ) / SPRY2 <-7-) cells, (c) (i) doubling times (ii) cell densities, (iii) pooled doubling times over all 4 passages and (iv) pooled cell size of bovine var. Wagyu adipose derived stem cell (ADSC) pools in suspension media containing no FBS and no growth factor. Average doubling time for the Ctrl is 37.2 h and 44.3 h for the PTEN <-7-) / SPRY2 <-7) cells. Average cell size for the Ctrl (PTEN <+7+VSPRY2 <+7+>) is 16.1 pm and 16.7 pm for PTEN<-7-) / SPRY2<-7-) cells.

[0101] Figure 17 illustrates the PTEN / SPRY2 double knockout can be combined with an HRAS G12D shows similar growth to HRAS G12D overexpression lines in an overgrowth study and both cell lines grow in media containing no FBS, no growth factors and no insulin: (a) (i) Viable cell densities, (ii) cell diameter) and (iii) cell viability of cells growing in suspension media without FBS and GF but containing insulin, (b) (i) Viable cell densities, (ii) cell diameter) and (iii) cell viability of cells growing in suspension media without FBS, GF, and insulin.

[0102] Figure 18 BAX and BAK1 can be edited efficiently in Wagyu muscle-derived cells and edits are retained over time, (a) the control cell line and (b) the BAX- / -BAK1- / - cell line demonstrate high knockout scores for P53, RB1 , NF2 and PTEN at both Day 3 and Day 10 post-editing.

[0103] Figure 19 Wagyu muscle-derived cells containing a BAX / BAK1 double knockout maintain peak cell density for longer and have a larger cell diameter in a suspension culture overgrowth study, (a) Cells containing a BAX / BAK1 double knockout maintained a peak viable cell density of around 4.6e6 cells / mL from day 6 to 9, whilst the cell density of the control cell line decreased during this time (b) Cells with a BAX / BAK1 double knockout had a larger diameter from day 4 to 10. (c) Average diameter over 10 days was significantly larger in BAX- / -BAK1- / - cells (14.0 pM) compared to the control cell line (13.4 pM).

[0104] Figure 20 Wagyu muscle-derived cells containing a BAX / BAK1 double knockout maintain peak cell density for longer and have a larger cell diameter in a suspension culture overgrowth study, (a) Cells containing a BAX / BAK1 double knockout maintained a peak viable cell density of around 5.3e6 cells / mL from day 5 to 7, whilst the cell density of the control cell line decreased during this time, (b) Cells with a BAX / BAK1 double knockout had a larger diameter from day 4 to 7. (c) Average diameter over 10 days was significantly larger in BAX- / -BAK1- / - cells (13.6 pM) compared to the control cell line (13.1 pM). (d) Annexin V staining demonstrated that there were fewer apoptotic cells present at Day 7 in BAX- / -BAK1- / - cells (22%) compared to the control cell line (53%).

[0105] Figure 21 Wagyu muscle-derived cells containing a BAX / BAK1 double knockout have improved apoptosis resistance in a suspension culture overgrowth study, (a) Cells containing a BAX / BAK1 double knockout maintained a viable cell density of around 4.9e6 cells / mL from day 5 to 7, whilst the cell density of the control cell line decreased during this time, (b) Cells with a BAX / BAK1 double knockout had a larger diameter, (c) Whilst viability was similar between the two cell lines (30% PI positive cells in the control cell line vs 23% in BAX- / -BAK1- / - cells); annexin v staining demonstrated that there were fewer apoptotic cells present at Day 7 in BAX^BAKI7- cells (16%) compared to the control cell line (45%).

[0106] Detailed description

[0107] The aspects of the invention will now be further described. In the following passages, different aspects are described. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary.

[0108] Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, pathology, oncology, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well-known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Green and Sambrook etal., Molecular Cloning: A Laboratory Manual, 4thed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012).

[0109] Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, cell biology and cell culturing, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for any cell culturing, genetic targeting, chemical syntheses, chemical analyses, and delivery.

[0110] Low cell doubling times and growth in suspension conditions are essential for large scale cultured meat processes. Somatic cells isolated from tissues / organs often used in food consumption (e.g. muscle, fat, fibroblasts) from agriculturally relevant species (e.g. pigs, cows, chickens) have a limited proliferation capacity when grown in vitro. Also, cells from animal tissue isolations tend to grow only in adherent conditions and the transfer into suspension conditions can lead to very slow cell growth (if any). Additionally, those cells are often grown in serum and / or growth factor containing media formulations, which are unsuitable for industrial cultivated meat production due to their associated costs and ethical concerns.

[0111] A method of culturing an animal cell

[0112] According to an aspect of the invention, there is provided a method of culturing an animal cell in serum fee or reduced serum media or serum free or reduced serum and growth factor free or reduced growth factor media. For example, according to an aspect of the invention, there is provided a method of culturing an animal cell comprising culturing the animal cell in a cell culture medium, wherein the cell culture medium is serum, exogenous growth factor, and / or insulin free or wherein the cell culture medium is reduced in serum, at least one exogenous growth factors, and / or insulin, and wherein the animal cell comprises a genetic modification in one or more of a RAS gene, the TP53 gene, the RB1 gene, the NF2 gene, the PTEN gene, the SPRY gene, the BAX gene and / or the BAK1 gene.

[0113] This is advantageous because the serum used in growth media is an animal derived product which can vary in components and consistency from batch to batch. This makes it an unsuitable element to include in food products and faces regulatory and ethical concerns over its use in cultured meat products. The same can be said for growth factors which should be kept to a minimum in human food products. Growth factors as well as serum are amongst the most expensive components of most culture media. For generating an economically viable food product, media costs must be kept to a minimum. Therefore, a method of reducing or abolishing the use of serum and / or growth factors is highly advantageous for the preparation of cultured meat products.

[0114] As used herein, the term "cell culture media" refers to solutions that contain factors and nutrients including, for example, growth factors, hormones, energy sources, amino acids, and organic and inorganic salts, which are used for the maintenance and growth of cells in ex vivo or in vitro culture. "Cell culture media" are often buffered to an approximately neutral pH (e.g., a pH of from about pH 6.6 to about pH 7.8) and can be supplemented.

[0115] As used herein, the term "cell culture medium supplement" refers to a component or mixture of components that are added to a cell culture medium, such as a basal cell culture medium to facilitate the maintenance and / or growth of a cell plated and / or seeded in such a supplemented cell culture medium. A cell culture medium supplement may be in a liquid for or may be lyophilized prior to use to promote the stability and shelf-life of the individual supplement components. Cell culture medium supplements include, for example, growth factors, hormones (e.g., the peptide hormone insulin), cytokines that promote the maintenance and / or growth of a cell, anti-oxidants, ribonucleotides, deoxyribonucleotides, anti-coagulants, sera, and / or antibiotics. Cell culture medium supplements are commonly added to a cell culture medium, such as a basal medium or a restrictive basal medium, immediately or shortly before use.

[0116] Cell culture medium supplements for culture of endothelial cells and endothelial progenitor cells can include one or more sera. As used herein, the terms "serum" and "sera" refer to the liquid portion of blood / plasma, which is typically also devoid of fibrinogen. Fetal bovine serum (FBS; a / k / a fetal calf serum (FCS)) is a common component of traditional EC growth media. It is usually added at low concentration (i.e., from about 2.5% (v / v) to about 5% (v / v)) to basal media that are specifically formulated for endothelial cells (EC) and at high concentrations (i.e., from about 10% (v / v) to about 30% (v / v)) to minimal basal media (e.g., DMEM / F-12 and M199). Reduced serum media can be defined as anything at 2.5% or less.

[0117] As used herein, the term "growth factor" refers to a protein or class of proteins, capable of stimulating cellular growth, cellular proliferation, cellular differentiation, and / or cellular maturation, such as, for example, EC proliferation and / or EPC differentiation. The term "growth factor" includes fragments of a protein capable of stimulating cellular growth, cellular proliferation, cellular differentiation, and / or cellular maturation. Examples of growth factors include, but are not limited to, basic fibroblast growth factor (FGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), nerve growth factor (NGF), platelet-derived growth factor-BB (PDGF-BB), long R3 insulin-like growth factor-1 (IGF-1), transforming growth factor beta (TGF-0), vascular endothelial growth factor (VEGF), bone morphogenetic proteins (BMP), and liver growth factor (LGF).

[0118] As used herein, the words “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 the DNA or RNA generated using nucleotide analogs. It can be single -stranded or double -stranded. Such nucleic acids or polynucleotides include, but are not limited to, coding sequences of structural genes, anti-sense sequences, and non-coding regulatory sequences that do not encode mRNAs or protein products. These terms also encompass a gene. The term “gene”, “allele” or “gene sequence” is used broadly to refer to a DNA nucleic acid associated with a biological function. Thus, genes may include introns and exons as in the genomic sequence, or may comprise only a coding sequence as in cDNAs, and / or may include cDNAs in combination with regulatory sequences. Thus, according to the various aspects of the invention, genomic DNA, cDNA or coding DNA may be used. In one aspect, the nucleic acid is cDNA or coding DNA. The terms “peptide”, “polypeptide” and “protein” are used interchangeably herein and refer to amino acids in a polymeric form of any length, linked together by peptide bonds. The term “allele” designates any of one or more alternative forms of a gene at a particular locus. Heterozygous alleles are two different alleles at the same locus. Homozygous alleles are two identical alleles at a particular locus. A wild type (wt) allele is a naturally occurring allele without a modification at the target locus.

[0119] It is described herein a method of culturing an animal cell comprising culturing the animal cell in a cell culture medium, wherein the cell culture medium is serum, exogenous growth factor, and / or insulin free or wherein the cell culture medium is reduced in serum, at least one exogenous growth factors, and / or insulin, and wherein the animal cell comprises a genetic modification in one or more of a RAS gene, the TP53 gene, the RB1 gene, the NF2 gene, the PTEN gene and / or a SPRY gene.

[0120] The media may comprise a reduced concentration of serum. The term reduced concentration may refer to below 2.5% serum, below 2.0% serum, below 1 .5% serum, below 1 .0% serum, 0.5% serum, or 0% serum. In one embodiment, the media is serum free. In one embodiment, the media may comprise at least 2.5%, 2.0.%, 1.5%, 1.0%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1 % or less serum. In one embodiment, the serum may be FBS, FCS, horse serum, rabbit serum, goat serum, new born calf serum, pig serum, or lamb / sheep serum. The skilled person would be aware of many other types of serum which could be used to supplement cell growth medium.

[0121] The media may comprise a reduced concentration of growth factors. The term reduced concentration of growth factors may refer to a media comprising at least one exogenous growth factors at a concentration of at least 20 ng / mL, 15 ng / mL, 10 ng / ml, 5 ng / ml, 4 ng / mL, 3 ng / mL, 2 ng / mL, 1 ng / mL or less. In one embodiment, the media is free from exogenous growth factors. In one embodiment, the media may comprise at least one exogenous growth factors at a concentration of 20 ng / mL or less. In one embodiment, the cell culture medium is serum, exogenous growth factor, and / or insulin free such that it comprises none of these supplements. In another embodiment, the cell culture medium may be free of one or more exogenous growth factor but still comprise one or more other exogenous growth factors.

[0122] In one embodiment, the media may be serum and growth factor free. In one embodiment, the media may be serum free and comprise reduced growth factor concentrations. In one embodiment, the media may be growth factor free and comprise reduced serum concentrations. In one embodiment, the media may comprise reduced concentrations of serum and growth factors.

[0123] In one embodiment, the cell culture medium comprises 100 pg / mL insulin or less.

[0124] In one embodiment, the animal is of an animal species suitable for human or animal consumption. For example, the animal is selected from a pig, bovine, poultry, sheep, goat, fish, Camelidae, Equidae, crustaceans or mollusc. In one embodiment, the animal cell is selected from one of the following cell types: myoblast, fibroblast, myofibroblast, adipose derived stem cell, epithelial cell, mesenchymal stem cell, satellite cell, iPSC, or hepatocyte. In one embodiment, the animal cell is a somatic cell. The animal cell may be selected from one of the following cell types: myoblast, fibroblast, myofibroblast, adipose derived stem cell, epithelial cell, mesenchymal stem cell, satellite cell, iPSC, or hepatocyte.

[0125] The genetic modification in animal cell can be targeted in a number of different ways, including at least one of:

[0126] 1) gene level modification by: a. knock-out or reduced activity / transcription / translation levels via editing in coding sequences, promoters, introns, regulatory regions; b. RNA-directed DNA methylation; or c. transcription activation or repression using CRISPRa or CRISPRi or similar target specific methods; d. knock-out or reduced activity / transcription / translation levels via undirected means, for example radiation or chemical mutagenesis; e. overexpression of an endogenous nucleotide sequence

[0127] 2) post-transcription level (post-transcriptional gene silencing) modification by: a. RNAi or siRNA to reduce translation of mRNA into protein; or b. site specific nucleases to modify or cleave mRNA, such as CRISPR / Cas13a;

[0128] 3) post-translational level (protein disruption or activation) modification by: a. inclusion of activity blocking / reducing or enhancing molecules, wherein the activity blocking / reducing or enhancing molecules are small molecules, antibodies, or the like; or b. inclusion of protein degrading ingredients, wherein the protein degrading ingredients are specialised proteases, exoproteases, or endoproteases. c. Enhancing or reducing of protein activity through integration of activating or reducing functional mutations in the corresponding gene sequences (endogenous or exogeneous).

[0129] Thus, invention provides manipulated primary animal cells that comprise modification of endogenous genes or expression of exogeneous nucleic acid constructs to overcome the cells’ need for growth factors and / or serum.

[0130] In one embodiment, the genetic modification may result in a decrease of 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%, or 95%. In one embodiment, the genetic modification may result in a decrease of the doubling time of the cell by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold or 100-fold. In one embodiment, the genetic modification increases cell size by at least about 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In one embodiment the genetic modification increases cell size by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6- fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold or 100-fold.

[0131] The term “decreasing” or “increasing” or the like is in relation to control or wild type cells which have not been genetically modified, or have not been genetically modified to influence expression levels of a gene of the invention.

[0132] In one embodiment, the genetic modification is a loss of function modification or leads to reduction in function. For example, the loss of function modification comprises a knock-out of the gene or loss of protein function.

[0133] In one embodiment, CRISPR may be used to knock out gene expression. In a further embodiment, single point mutation or multiple mutations may be used to knock out gene expression. In a yet further embodiment, the CRISPR / Cas9 or CRISPR / Cas13a systems may be used to knock out gene expression.

[0134] According to one embodiment, the expression of the gene may be modified in an animal cell by at least one small molecule or at least one RNAi. In one embodiment, the RNAi may be shRNA or siRNA.

[0135] When double stranded RNAs are processed by an Rnase Ill-like protein known as Dicer mRNA can be silenced and protein activity lowered or abolished within a cell. 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 the long double stranded RNAs creates 21-24 nucleotide double stranded siRNAs with 2 base 3’ overhangs and a 5’ phosphate and 3’ hydroxyl group. The resulting siRNA duplexes are then incorporated into the effector complex known as RNA-induced silencing complex (RISC), where the antisense or guide strand of the siRNA guides RISC to recognize and cleave target mRNA sequences (Elbashir et al 2001) upon adenosine-triphosphate (ATP)-dependent unwinding of the double-stranded siRNA molecule through an RNA helicase activity (Nykanen et al 2001). The catalytic activity of RISC, which leads to mRNA degradation, is mediated by the endonuclease Argonaute 2 (AGO2) (Liu et al 2004; Song et al 2004). AGO2 belongs to the highly conserved Argonaute family of proteins. Argonaute proteins are GO Kda highly basic proteins that contain two common domains, namely PIWI and PAZ domains (Cerutti et al 2000). The PIWI domain is crucial for the interaction with Dicer and contains the nuclease activity responsible for the cleavage of mRNAs. AGO2 uses one strand of the siRNA duplex as a guide to find messenger RNAs containing complementary sequences and cleaves the phosphodiester backbone between bases 10 and 11 relative to the guide strand’s 5’ end (Elbashir et al 2001). An important step during the activation of RISC is the cleavage of the sense or passenger strand by AGO2, removing this strand from the complex (Rand et al 2005). Once the mRNA has been cleaved, due to the presence of unprotected RNA ends in the fragments the mRNA is further cleaved and degraded by intracellular nucleases and will no longer be translated into proteins. This leads to reduction of specific mRNA molecules and the corresponding proteins. It is possible to exploit this native mechanism for gene silencing with the purpose of regulating any gene(s) of choice.

[0136] Many studies have been published describing how to optimise siRNA, for example WO02 / 44321 , (Walton SP et al 2010, and Chang Cl et al 2011) the contents of which are incorporated herein by reference.

[0137] Godinho and Khvorova 2019 describes commonly used methods of delivering RNAi into cells. Examples of materials used as non-viral vectors, nanocarriers or in formulated nanosystems and ligands used for conjugation are described. Additional examples are provided in references included therein. Nano particles are often used to introduce RNAi into cells and cationic lipids (e.g. D-Lin-MC3- DMA), polymers (e.g. cyclodextrin-based polymers and biocollagen), polypeptides, and exosomes are all examples of biomaterials which can be used to transport RNAi into the cytoplasm of target cells.

[0138] The creation and delivery of RNAi are further described in US6,506,559 and W02007045930 the contents of which are incorporated herein by reference.

[0139] In some embodiments, post-translational Inactivation (protein disruption) can be used to inhibit the activity of the protein expressed from the gene. In one embodiment, activity blocking / reducing molecules can inhibit protein activity. For example, small molecules can inhibit protein activity.

[0140] In some embodiments, protein expressed from the gene may be degraded using protein degrading ingredients. Examples include i) specialised proteases, e.g. a calcium-dependent cysteine protease, such as calpain, ii) exoproteases, or iii) endoproteases.

[0141] According to the various aspects of the invention, the modification can be in the promoter region or in the coding region of the one of more genes that is / are targeted. The cell is therefore genetically manipulated I engineered.

[0142] In one embodiment of the aspects of the invention, the modified cell is a primary cell. In another embodiment of the aspects of the invention, the modified cell is a somatic cell. Any somatic cell suitable for use in cellular agriculture, that is the production of animal-sourced foods from cell culture, is within the scope of the invention. For example, the cell may be a fat or muscle cell. For example, the cell may be selected from one or more of the following cell types: myoblast, fibroblast, myofibroblast, adipose derived stem cell, epithelial cell, mesenchymal stem cell, satellite cell, iPSC, or hepatocytes.

[0143] The terms “animal” and “non-human animal” with reference to animals and cells derived therefrom are used herein interchangeably and refer only to cells of non-human-animals. Cells for use in the invention may be of any other animal origin. However, the cells are not human cells. Cells suitable for use in cellular agriculture are preferably non-human animal cells that provide a source of any dietary protein, fat and / or carbohydrate.

[0144] The cells are cells of non-human animals that are suitable for human and animal consumption. These include animals such as non-human mammals, birds, fish, crustaceans, molluscs, reptiles, amphibians, or insects. Exemplary non-human mammals include those in the genera Bovinae, Camelidae, Canidae, Caprae, Cervidae, Felidae, Equidae, Lagomorphs, Macropodidae, Oves, Rodents, or Suidae. The cells may be cells of any livestock or poultry. The cells may be porcine, bovine (e.g. cattle), ovine, caprine, avine, or piscine. The cell may be shrimp, prawn, crab, crayfish, and / or lobster. In one embodiment, the animal is a pig or bovine (e.g. cattle).

[0145] The animal used in various aspects of the invention may be of an animal species used in agriculture. An animal species used in is an animal farmed for human. Such animals are listed above. In a preferred embodiment, they include pig, bovine (e.g. cattle), poultry (e.g. chicken, turkey, duck, geese), sheep, goat, Equidae, Camelidae, fish, crustaceans or mollusc.

[0146] The doubling time of a cell line is the average time it takes for a population of the cells to double in number as a result of cell cycle progression and subsequent division. Therefore, removing cell cycle checkpoint inhibition of the cell cycle decreases the time required for one cell to undergo mitosis and form two new daughter cells. When applied to a whole population of cells of the cell line, this modification reduces the doubling time of said cell line and means the cells are quick to expand and better suited for use in cellular agriculture.

[0147] The term “genetic modification” relates to a modification that alters expression of the gene that is targeted or functional activity of the gene product, i.e. the gene. The genetic modification may result in a loss of function, for example by creating a knock-out. To create a loss of function / knock-out, a mutation may be introduced in the coding sequence which renders the expressed protein non-functional (e.g. an amino acid substitution, deletion or addition / insertion) or creates a premature stop codon / prevents expression of a functional protein.

[0148] Examples of loss of function mutations are described herein. However, any mutation that results in a dominant loss of function as described herein is encompassed within the scope of the invention. As used herein, “dominant” also encompasses “semi-dominant” or “partially dominant”. Therefore, the mutant allele may be fully dominant, partially dominant or semi-dominant. Preferably, the mutant allele is fully dominant. A loss of function mutation includes a knock-out modification or any other modification that causes an amino acid substitution or change wherein the substitution or change causes the resulting protein to lack a specific function or causes a reduction in the activity of said protein or prevents expression of the protein. Preferably, both alleles of the gene are knocked out.

[0149] A knock-out modification or mutation may eliminate at least partially the specific endogenous nucleic acid sequence from the genomic DNA of the cell that codes for the protein of interest. By eliminating the corresponding nucleic acid sequence, the protein can no longer be synthesised by the cellular machinery. In some embodiment, a knock-out modification occurs from the introduction of an indel (insertion and / or deletion event) that results in a change of the native amino acid composition of the resulting protein, often this takes the form of a frameshifting mutation.

[0150] Examples of gain of function are also described herein. In some embodiment, a change in the gene sequence might lead to a change to the native amino acid composition of the resulting protein, which increases the activity of said protein. In a further embodiment, changes in the amino acid sequence of a protein might add or remove regulatory regions of a protein, such as, but not limiting to, phosphorylation, acetylation and ubiquitination sites. In a further embodiment, changes in the amino acid sequence of a protein might stabilize or destabilize intermediate states of its enzymatic reaction substrates and products. In a further embodiment, changes in the amino acid sequence of a protein might lock it in a permanent “on”-state. In a further embodiment, changes in regulatory gene sequences might increase transcription of a gene and thereby protein amount and the total activity of that protein per cell. In a further embodiment, changes in the amino acid sequence of a protein might influence its’ interactions with other proteins. In a further embodiment, gain of function changes are introduced in a targeted manner by knock-in of specific nucleotides which lead to desired amino acid changes on protein level or desired nucleotide exchanges in regulatory gene regions. In a further embodiment, gain of function mutations are introduced into cells by addition of exogeneous or endogenous nucleotide sequences encoding for the desired protein. In a further embodiment, gain of function mutations are introduced into cells by addition of exogeneous nucleotide sequences encoding for specific promoter sequences to drive gene expression.

[0151] In one embodiment, the genetic modification to the RAS gene is an overexpression and / or hyperactivation of the gene or gene product. For example, the RAS gene is HRAS, NRAS, or KRAS.

[0152] An amino acid substitution is affected by alterations in a nucleic acid sequence that results in the production of a different amino acid at a given site. This modification may affect the functional properties and / or activity of the encoded polypeptide or it may not affect the functional properties of the encoded polypeptide (conservative substitution). Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products. Non-conservative substitution leads to an encoded protein which does not retain the same functional properties and / or activity of the non-modified protein.

[0153] Sequence identity is commonly defined with reference to the algorithm GAP (Wisconsin GCG package, Accelerys Inc, San Diego USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences, maximising the number of matches and minimising the number of gaps. Generally, default parameters are used, with a gap creation penalty typically equalling 12 and a gap extension penalty equalling 4. Use of GAP may be preferred but other algorithms may be used, e.g. BLAST, or the Smith- Waterman algorithm, or the TBLASTN program, of, generally employing default parameters. In particular, the psi-Blast algorithm may be used. Sequence identity may be defined using the Bioedit, ClustalW algorithm. Alignments can be performed using Snapgene and based on MUSCLE (Multiple Sequence Comparison by Log-Expectation) algorithms. Sanger Sequencing of PCR amplicons and analysis of the sequencing results using the ICE analysis tool (ice.synthego.com) were also used to confirm sequence identity.

[0154] In one embodiment, the modification is introduced using targeted genome modification and / or a rare- cutting endonuclease, for example a meganuclease, TALEN, ZFN or CRISPR / Cas9.

[0155] Genome editing techniques have emerged as alternative methods to conventional mutagenesis methods (such as physical and chemical mutagenesis) or methods using the expression of transgenes in animal cells to produce mutant animal cells with improved phenotypes that are important in cellular research and cellular agriculture. These techniques employ sequence -specific nucleases (SSNs) including meganucleases, zinc finger nucleases (ZFNs), transcription activator -like effector nucleases (TALENs), and the RNA -guided nuclease Cas9 (CRISPR / Cas9), which generate targeted DNA double -strand breaks (DSBs), which are then repaired mainly by either error -prone non-homologous end joining (NHEJ) or high- fidelity homologous recombination (HR).

[0156] As explained in detail below, mutations according to the various aspects of the invention can be introduced into animal cells using targeted genome modification based on such editing techniques.

[0157] In another aspect, the invention also relates to a method for modifying the expression or function of one or more genes in a non-human animal wherein the gene is associated with cellular signalling pathways. In a preferred embodiment, the animal is an animal suitable for human or animal consumption, for example used in agriculture. In an embodiment, the method comprises introducing a mutation into the one or more genes in the animal cell.

[0158] In another aspect, the invention relates to a method of producing a modified non-human animal cell described herein, wherein the method comprises introducing a genetic modification in one or more genes associated with cellular signalling pathways. In a preferred embodiment, the animal is an animal used in agriculture.

[0159] In yet another aspect the invention provides a modified animal cell having a genetic modification. The modified animal cell may be an immortalised animal cell. In a further embodiment, the immortalised animal cell may have been immortalised using targeted genome modification. In other embodiments, the immortalised animal cell may have been immortalised using randomised mutagenesis. In yet further embodiments, the immortalised animal cell may have been immortalised using radiation or chemical mutagenesis. In yet further embodiments, the immortalised animal cell may have been spontaneously immortalised.

[0160] In some embodiments, the genetic modification in the gene may be created using targeted genome modification. In other embodiments, the genetic modification in the gene may be created using randomised mutagenesis. In yet further embodiments, the genetic modification in the gene may have occurred spontaneously.

[0161] In all aspects of the invention the animal is not human. Animals that can be used, in particular agriculturally relevant animals, are listed herein. modification of animal cells

[0162] Targeted genome modification or targeted genome editing is a genome engineering technique that uses targeted DNA double-strand breaks (DSBs) to stimulate genome editing through homologous recombination (HR)-mediated recombination events. To achieve effective genome editing via introduction of site-specific DNA DSBs, four major 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 endonucleases (SSN), for example TALENs, transcription activator-like effectors (TALEs) from Xanthomonas bacteria, 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 would be well known to the skilled person in the art. Meganuclease, 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 consist of individual modules targeting 3 or 1 nucleotides (nt) of DNA, respectively. ZFs and TALEs can be assembled in desired combinations and attached to the nuclease domain of Fokl to direct nucleolytic activity toward specific genomic loci.

[0163] Upon delivery into host cells via the bacterial type III secretion system, TAL effectors enter the nucleus, bind to effector-specific sequences in host gene promoters 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. The majority of naturally occurring TAL effectors examined have between 12 and 27 full repeats.

[0164] These repeats only differ from each other by two adjacent amino acids, their repeat- variable di-residue (RVD). The RVD determines which single nucleotide the TAL effector will recognize: one RVD corresponds to one nucleotide, with the four most common RVDs each preferentially associating with one of the four bases. Naturally occurring recognition sites are uniformly preceded by a T that is required for TAL effector activity. TAL effectors can be fused to the catalytic domain of the Fokl nuclease to create a TAL effector nuclease (TALEN) which makes 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, for example in US 8,440,431 , US 8,440, 432 and US 8,450,471 . Customized 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 cleave outside their recognition sites to create unique 4 bp overhangs. Cloning is expedited by digesting and ligating in the same reaction mixture because correct assembly eliminates the enzyme recognition site. Assembly of a custom TALEN or TAL effector construct and involves two steps: (i) assembly of repeat modules into intermediary arrays of 1-10 repeats and (ii) joining of the intermediary arrays into a backbone to make the final construct.

[0165] Another genome editing method that can be used according to the various aspects of the invention is CRISPR. The use of this technology in genome editing is well described in the art, for example in US 8,697,359. In short, CRISPR is a microbial nuclease system involved in defence against invading phages and plasmids. CRISPR loci in microbial hosts contain a combination of CRISPR--associated (Cas) genes as well as non-coding RNA elements capable of programming the specificity of the CRISPR-mediated nucleic acid cleavage. One key feature of each CRISPR locus is the presence of an array of repetitive sequences (direct repeats) interspaced by short stretches of non-repetitive sequences (spacers). The non-coding CRISPR array is transcribed and cleaved within direct repeats into short crRNAs containing individual spacer sequences, which direct Cas nucleases to the target site (protospacer).

[0166] By “crRNA” or CRISPR RNA is meant the sequence of RNA that contains the protospacer element and additional nucleotides that are complementary to the tracrRNA. By “tracrRNA” (transactivating RNA) is meant the sequence of RNA that hybridises to the crRNA and binds a CRISPR enzyme, such as Cas9 thereby activating the nuclease complex to introduce double-stranded breaks at specific sites within the genomic sequence of at least one nucleic acid or promoter sequence of the one or more genes. By “protospacer element” is meant the portion of crRNA (or sgRNA) that is complementary to the genomic DNA target sequence, usually around 20 nucleotides in length. This may also be known as a spacer or targeting sequence. By “sgRNA” (single-guide RNA) is meant the combination of tracrRNA and crRNA in a single RNA molecule, preferably also including a linker loop (that links the tracrRNA and crRNA into a single molecule). “sgRNA” may also be referred to as “gRNA” and in the present context, the terms are interchangeable. The sgRNA or gRNA provide both targeting specificity and scaffolding / binding ability for a Cas nuclease. A gRNA may refer to a dual RNA molecule comprising a crRNA molecule and a tracrRNA molecule.

[0167] The Type II CRISPR is one of the most well characterized systems and carries out targeted DNA double-strand breaks in four sequential steps. First, two non-coding RNA, the pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus. Second, tracrRNA hybridizes to the repeat regions of the pre-crRNA and mediates the processing of pre-crRNA into mature crRNAs containing individual spacer sequences. Third, the mature crRNA: tracrRNA complex directs 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), an additional requirement for target recognition. Finally, Cas9 mediates cleavage of target DNA to create a double-stranded break within the protospacer. Cas9 is thus the hallmark protein of the type I CRISPR-Cas system, and a large monomeric DNA nuclease guided to a DNA target sequence adjacent to the PAM sequence motif by a complex of two noncoding 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 whereas the RuvC-like domain cleaves the non-complementary strand and, as a result, a blunt cut is introduced in the target DNA. Heterologous expression of Cas9 together with a guide RNA (gRNA) also called single guide RNA (sgRNA) can introduce site-specific double strand breaks (DSBs) into genomic DNA of live cells from various organisms. For applications in eukaryotic organisms, codon optimized versions of Cas9, which is originally from the bacterium Streptococcus pyogenes, have been used.

[0168] Synthetic CRISPR systems typically consist of two components, the gRNA and a non-specific CRISPR-associated endonuclease and can be used to generate knock-out cells or animals by co-expressing a gRNA specific to the gene to be targeted and capable of association with the endonuclease Cas9. Notably, the gRNA is an artificial molecule comprising one domain interacting with the Cas or any other CRISPR effector protein or a variant or catalytically active fragment thereof and another domain interacting with the target nucleic acid of interest and thus representing a synthetic fusion of crRNA and tracrRNA. The genomic target can be any 20 nucleotide DNA sequence, provided that the target is present immediately upstream of a PAM sequence. The PAM sequence is of outstanding importance for target binding and the exact sequence is dependent upon the species of Cas9.

[0169] The PAM sequence for the Cas9 from Streptococcus pyogenes has been described to be “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 sequence for Cas9 from Staphylococcus aureus is “NNGRRT” or “NNGRR(N)”. Further variant CRISPR / Cas9 systems are known. Thus, a Neisseria meningitidis Cas9 cleaves at the PAM sequence NNNNGATT. A Streptococcus thermophilus Cas9 cleaves at the PAM sequence NNAGAAW. Recently, a further PAM motif NNNNRYAC has been described for a CRISPR system of Campylobacter (WO 2016 / 021973). For Cpf1 nucleases, e.g. Cas12a, it has been described that the Cpf1-crRNA complex, without a tracrRNA, efficiently recognize and cleave target DNA proceeded by a short T-rich PAM in contrast to the commonly G-rich PAMs recognized by Cas9 systems. Furthermore, by using modified CRISPR polypeptides, specific single-stranded breaks can be obtained. The combined use of Cas nickases with various recombinant gRNAs can also induce highly specific DNA double-stranded breaks by means of double DNA nicking. By using two gRNAs, moreover, the specificity of the DNA binding and thus the DNA cleavage can be optimized. Further CRISPR effectors like CasX and CasY effectors originally described for bacteria, are meanwhile available and represent further effectors, which can be used for genome engineering purposes (Burstein et al., “New CRISPR-Cas systems from uncultivated microbes”, Nature, 2017, 542, 237-241).

[0170] Once expressed, the Cas9 protein and the gRNA form a ribonucleoprotein complex through interactions between the gRNA “scaffold” domain and surface-exposed positively-charged grooves on Cas9. Cas9 undergoes a conformational change upon gRNA binding that shifts the molecule from an inactive, non-DNA binding conformation, into an active DNA-binding conformation. Importantly, the “spacer” sequence of the gRNA remains free to interact with target DNA. The Cas9-gRNA complex will bind any genomic sequence with a PAM, but the extent to which the gRNA spacer matches the target DNA determines whether Cas9 will cut. Once the Cas9-gRNA complex binds a putative DNA target, a “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 a 3' to 5' direction (relative to the polarity of the gRNA).

[0171] CRISPR / Cas9 and likewise CRISPRZCpfl and other CRISPR systems are highly specific when gRNAs are designed correctly, but especially specificity is still a major concern, particularly for clinical uses based on the CRISPR technology. The specificity of the CRISPR system is determined in large part by how specific the gRNA targeting sequence is for the genomic target compared to the rest of the genome. The sgRNA is a synthetic RNA chimera created by fusing crRNA with tracrRNA. The sgRNA guide sequence located at its 5' end confers DNA target specificity. Therefore, by modifying the guide sequence, it is possible to create sgRNAs with different target specificities. The canonical length of the guide sequence is 20 bp.

[0172] Thus, as used herein, the term “guide RNA” relates to a synthetic fusion of two RNA molecules, a crRNA (CRISPR RNA) comprising a variable targeting domain, and a tracrRNA. In one embodiment, the guide RNA comprises a variable targeting domain of 12 to 30 nucleotide sequences and a RNA fragment that can interact with a Cas endonuclease. sgRNAs suitable for use in the methods of the invention are described below. As used herein, the term “guide polynucleotide”, relates to a polynucleotide sequence that can form a complex with a Cas endonuclease and enables 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 (a RNA-DNA combination 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 to a cholesterol molecule, linkage to a polyethylene glycol molecule, linkage to a spacer 18 (hexaethylene glycol chain) molecule, or 5' to 3' covalent linkage resulting in circularization. A guide polynucleotide that solely comprises ribonucleic acids is also contemplated.

[0173] 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 a polynucleotide sequence in the genome (including mitochondrial DNA) of a cell at which a double-strand break is induced in the cell genome by a Cas endonuclease. The target site can be an endogenous site in the genome, or alternatively, or the target site can be found in a heterologous genomic location compared to where it occurs in nature. As used herein, terms “endogenous target sequence” and “native target sequence” are used interchangeably herein to refer to a target sequence that is endogenous or native to the genome and is at the endogenous or native position of that target sequence in the genome.

[0174] The length of the target site can vary, and includes, for example, target sites that are at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides in length. It is further possible that the target site can be palindromic, that is, the sequence on one strand reads the same in the opposite direction on the complementary strand. The nick / cleavage site can be within the target sequence or the nick / cleavage site could be outside of the target sequence. In another variation, the cleavage could occur at nucleotide positions immediately opposite each other to produce a blunt end cut or, in other cases, the incisions could be staggered to produce single stranded overhangs, also called “sticky ends”, which can be either 5' overhangs, or 3' overhangs.

[0175] In one embodiment, the Cas endonuclease gene is a Cas9 endonuclease, such as but not limited to, Cas9 genes listed in W02007 / 025097 incorporated herein by reference. In another embodiment, the Cas endonuclease gene is animal optimized Cas9 endonuclease. In one embodiment, the Cas endonuclease gene is an animal codon optimized streptococcus pyogenes Cas9 gene that can recognize any genomic sequence of the form N(12--30)NGG can in principle be targeted.

[0176] In one embodiment, the Cas endonuclease is introduced directly into a cell by any method known in the art, for example, but not limited to transient introduction methods and / or transfection.

[0177] Cas9 expression plasmids for use in the methods of the invention can be constructed as described in the art.

[0178] In one embodiment, targeted genome modification according to the various aspects of the invention comprises the use of a rare cutting endonuclease, for example a meganuclease, TALEN, ZFN or CRISPR / Cas; e.g. CRISPR / Cas9. Rare cutting endonucleases / sequence specific endonucleases are naturally or engineered proteins having endonuclease activity and are target specific. These bind to nucleic acid target sequences which have a recognition sequence typically 12-40 bp in length. In one embodiment, the SSN is selected from a TALEN. In another embodiment, the SSN is selected from CRISPR / Cas9. This is described in more detail below.

[0179] In one embodiment, the step of introducing a mutation comprises transfecting a population of animal cells with DNA binding protein targeted to the gene. In one embodiment, the method comprises contacting a population of cells with one or more rare-cutting endonucleases; e.g. meganucleases, ZFN, TALEN, or CRISPR / Cas9, targeted to the gene.

[0180] The method may further comprise the steps of selecting, from said population, a cell in which the gene sequence has been modified and regenerating said selected animal cell.

[0181] In an embodiment, the method comprises the use of CRISPR / Cas9. In this embodiment, the method therefore comprises introducing and co-expressing in an animal cell Cas9 and sgRNA targeted to the gene sequences and screening for induced targeted mutations in the gene.

[0182] Cas9 and sgRNA may be comprised in a single or two expression vectors. The target sequence is the gene nucleic acid sequence as shown herein.

[0183] In one embodiment, screening for CRISPR-induced targeted mutations in the gene comprises obtaining a DNA sample from a transfected / transduced animal cell and carrying out DNA amplification and optionally restriction enzyme digestion to detect a mutation in the gene.

[0184] In one embodiment, the restriction enzyme is mismatch-sensitive T7 endonuclease. T7E1 is an enzyme that is specific to heteroduplex DNA caused by genome editing. PCR fragments amplified from the transfected / transduced animal cells are then assessed using a gel electrophoresis based assay. In a further step, the presence of the mutation may be confirmed by sequencing the gene and analysing the sequencing histograms using programs known to the skilled person in the art, such as the ICE analysis tool or the TIDE analysis tool. Genomic DNA (i.e. wt and mutant) can be prepared from each sample, and DNA fragments encompassing each target site are amplified by PCR. The PCR products are digested by restriction enzymes as the target locus includes a restriction enzyme site. The restriction enzyme site is destroyed by CRISPR- or TALEN-induced mutations by NHEJ or HR, thus the mutant amplicons are resistant to restriction enzyme digestion, and result in uncleaved bands. Alternatively, the PCR products are digested by T7E1 (cleaved DNA produced by T7E1 enzyme that is specific to heteroduplex DNA caused by genome editing) and visualized by agarose gel electrophoresis. In a further step, they are sequenced.

[0185] In one embodiment, the method uses the sgRNA (and template, synthetic single-strand DNA oligonucleotides (ssDNA oligos) or donor DNA) constructs defined in detail below to introduce a targeted SNP or mutation, in particular one of the substitutions described herein into the gene and / or promoter. The introduction of a template DNA strand, following a sgRNA-mediated SNP in the double-stranded DNA, can be used to produce a specific targeted mutation (i.e. a SNP) in the gene using homology directed repair. Synthetic single-strand DNA oligonucleotides (ssDNA oligos) or DNA plasmid donor templates can be used for precise genomic modification with the homology-directed repair (HDR) pathway. Homologous recombination is the exchange of DNA sequence information through the use of sequence homology. Homology-directed repair (HDR) is a process of homologous recombination where a DNA template is used to provide the homology necessary for precise repair of a double-strand break (DSB). CRISPR guide RNAs program the Cas9 nuclease to cut genomic DNA at a specific location. Once the double-strand break (DSB) occurs, the mammalian cell utilizes endogenous mechanisms to repair the DSB. In the presence of a donor DNA, either a ssDNA oligo or a plasmid donor, the DSB can be repaired precisely using HDR resulting in a desired genomic alteration (insertion, removal, or replacement).

[0186] Single-strand DNA donor oligos are delivered into a cell to insert or change short sequences (SNPs, amino acid substitutions, epitope tags, etc.) of DNA in the endogenous genomic target region.

[0187] A “donor sequence” is a nucleic acid sequence that contains all the necessary elements to introduce the specific substitution into a target sequence, preferably using homology-directed repair (HDR). In one embodiment, the donor sequence comprises a repair template sequence for introduction of at least one SNP. Preferably the repair template sequence is flanked by at least one, preferably a left and right arm, that are identical to the target sequence. The methods above use animal cell in which an expression vector has been introduced comprising a sequence-specific nucleases into an animal cell to target the RAS gene, the TP53 gene, the RB1 gene, the RB1 gene, the NF2 gene, the PTEN gene, SPRY gene, the BAK1 and / or the BAX nucleic acid sequence. The term "introduction” or "transfected / transduced” as referred to herein encompasses the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer.

[0188] Advantageously, any of several transfection / transduction methods may be used to introduce the gene of interest into a suitable cell. The methods described for the transfection / transduction of animal cells may be utilized for transient or for stable transfection / transduction. transfection / transduction methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the animal cell, particle bombardment as described in the examples, transfection / transduction using viruses or microinjection. Methods may be selected from, microinjection into animal material, DNA or RNA-coated particle bombardment, infection with (non-integrative or integrative) viruses and the like.

[0189] Following DNA transfer and regeneration, putatively transformed animal cells may also be evaluated, for instance using Southern analysis, for the presence of the gene of interest, copy number and / or genomic organisation. Alternatively or additionally, expression levels of the newly introduced DNA may be monitored using Northern and / or Western analysis, both techniques being well known to persons having ordinary skill in the art.

[0190] The sequence-specific nucleases may also be introduced into an animal cell as part of an expression vector. The vector may contain one or more replication systems which allow it to replicate in host cells. Self-replicating vectors include plasmids, cosmids and virus vectors. Alternatively, the vector may be an integrating vector which allows the integration into the host cell’s chromosome of the DNA sequence. The vector desirably also has unique restriction sites for the insertion of DNA sequences. If a vector does not have unique restriction sites it may be modified to introduce or eliminate restriction sites to make it more suitable for further manipulation. Vectors suitable for use in expressing the nucleic acids, are known to the skilled person and a non-limiting example is 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 the nucleic acids include, but are not limited to PGK, CMV, EF1 a, CAG, SV40 and Ubc.

[0191] In an embodiment of the invention the modification is made to the promoter region or coding region of the one or more genes.

[0192] Genetic modifications The animal cell may comprise a genetic modification in one or more of a RAS gene, the TP53 gene, the RB1 gene, the NF2 gene, the PTEN gene, a SPRY gene, the BAX gene and / or the BaAK1 gene. In one embodiment, the SPRY gene is selected from SPRY1 SPRY2, SPRY3, and / or SPRY4 or a combination thereof.

[0193] The genetic modification in the TP53 gene, the RB1 gene, the NF2 gene, the PTEN gene, SPRY gene, the BAX gene and / or the BAK1 gene is a loss of function modification. The loss of function modification comprises a knock-out of the gene. For example, the genetic modification in the TP53 gene, the RB1 gene, the NF2 gene, the PTEN gene, a SPRY gene, the BAX gene and / or the BAK1 gene is a knock out genetic modification in one or both alleles.

[0194] The genetic modification may be in a RAS gene. For example, the RAS gene is HRAS, NRAS, or KRAS. The genetic modification to the RAS gene may be an overexpression and / or hyperactivation of the gene or gene product.

[0195] The genetic modification to a RAS gene, the TP53 gene, the RB1 gene, the NF2 gene, the PTEN gene, a SPRY gene, the BAX gene and / or the BAK1 gene may be made individually or in any combination. In one embodiment, the genetic modification may be a single point mutation or multiple mutations to knock out gene expression, e.g. for the RB1, TP53, NF2 genes, the PTEN gene, a SPRY gene, the BAX gene and / or the BAK1 gene or the genetic modification may be a single point mutation or multiple mutations or integration of a cis- or transgene to enhance gene expression and / or protein activity, e.g. for the RAS gene.

[0196] The invention therefore may relate to an animal cell with reduced protein expression from the TP53 gene, the RB1 gene, the NF2 gene, the PTEN gene, the SPRY gene, the BAX gene and / or the BaAK1 gene and / or increased protein expression of the RAS gene.

[0197] The genetic modification(s) may be selected from any of those listed in Table 1 .

[0198] Table 1

[0199]

[0200] In some embodiments the animal cell has a single genetic modification in each of the genes. In one embodiment the animal cell has at least one genetic modification in each of the genes. In one embodiment the animal cell has multiple a genetic modification in each of the genes.

[0201] In one embodiment, the animal cell comprises a genetic modification in the RB1 gene, the TP53 gene, the NF2 gene, a RAS gene, the PTEN gene, the SPRY gene, the BAX gene and / or the BAK1 gene and wherein the animal is of an animal species suitable for human or animal consumption.

[0202] In one embodiment, the RAS gene is HRAS, NRAS, or KRAS. In one embodiment, the RAS gene is HRAS.

[0203] Preferably, the modified cell may be a porcine cell or bovine cell and the targeted gene is selected from RB1, TP53, NF2, PTEN, SPRY, BAX, BAK1 and / or HRAS, NRAS, or KRAS. Further, examples for suitable genetic modifications in porcine and bovine species are set out in Applicant’s own PCT / GB2023 / 052528 and PCT / GB2023 / 053374, which are hereby incorporated by reference.

[0204] A skilled person would know that for the manipulation of other animal cells from animal species suitable for human or animal consumption, e.g. suitable for human consumption, e.g. suitable for animal consumption, e.g. used in agriculture, e.g. as listed herein, the equivalent orthologue gene specific to the non-human animal species targeted is to be genetically modified. Suitable gene sequences can be identified from public databases. A skilled person would also be able to identify suitable sequences using standard methods in the art to identify homologs and orthologs, for example based on sequence identity with the pig sequences.

[0205] In a further embodiment of the invention, the gene is RAS and the modification is a hyperactivation modification. In one embodiment, the resulting modifications in the RAS protein keep it in a constantly active state. In one embodiment, activating modifications reduce GTP hydrolysis. The RAS gene may be selected from any one of HRAS, NRAS, or KRAS (isoform A or isoform B). In a related embodiment of the invention, the hyperactivation modification comprises one or more amino acid substitutions in the protein. The RAS gene may be a porcine RAS gene. The RAS gene may be a bovine RAS gene. In one embodiment, gain of function mutations are introduced into the RAS genes using targeted nucleases or derivations thereof. In another embodiment, gain of function versions of the RAS proteins are introduced as exogenous nucleotides into the cells. In one embodiment, the gain of function mutations are introduced via random mutagenesis, including but not limited to chemical mutagenesis. In one embodiment, the gain of function mutations are introduced by spontaneous mutations. In another embodiment, RAS activity is increased by perturbing other regulators of RAS activity.

[0206] For example, in one embodiment, the animal cell may be a sus scrofa var. Largewhite in which the HRAS gene comprises a hyperactivation modification and has a nucleic acid sequence according to SEQ ID NO: 26 and a protein sequence according to SEQ ID NO: 27.

[0207] In one embodiment, the animal cell may be a sus scrofa var. Largewhite in which the NRAS gene comprises a hyperactivation modification and has a nucleic acid sequence according to SEQ ID NO: 28 and a protein sequence according to SEQ ID NO: 29.

[0208] In one embodiment, the animal cell may be a sus scrofa var. Largewhite in which the KRAS gene comprises a hyperactivation modification and has a nucleic acid sequence according to SEQ ID NO: 30 and a protein sequence according to SEQ ID NO: 31 .

[0209] Further examples are described in detail in Applicant’s own PCT / GB2023 / 052528 and PCT / GB2023 / 053374, which are hereby incorporated by reference. Cultivated meat and methods

[0210] In another aspect, the invention provides a method of producing cultivated meat / a cultivated meat product I food product comprising culturing a modified cell according to any previous embodiments of the invention. In a related embodiment, the method comprises carrying out continuous or batch culture of the modified cell.

[0211] The term "cultivated meat” is used herein to describe meat grown from in vitro animal cell culture distinguished from meat of slaughtered animals. Additional terms that may be used in the Art to describe meat grown from in vitro animal cell culture include cultured meat, cell-grown meat, clean meat, lab- 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 manmade meat. The phrases “cellbased 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 such phrases are interchangeably used herein, and refer to the meat that is generated in vitro, starting with cells in culture, and that method which does not involve the slaughter of an animal in order to directly obtain meat from that animal for dietary consumption. The modified cells of the invention may be suitable for human and / or non-human consumption. In some embodiments, the cell-based meat is suitable for consumption by animals, such as domesticated animals. Accordingly, the cellular biomass herein supports the growth of “pet food”, e.g. dog food, cat food, and the like.

[0212] In one embodiment, the invention is a cultured animal cell comprising at least one of the aforementioned genetic modifications and wherein the animal is of 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 animal cell. In a yet further embodiment, the cultured animal cell is not a treatment for cancer. In a still further embodiment, the invention does not relate to treatments for cancer or to products or genetic modifications for use in the treatment of cancer.

[0213] Batch culture refers to culturing cells in a closed system whereby the culture of cells is carried out for a defined period of time or until a defined criteria is met. Once this criteria or time is met the culture is stopped, the cells harvested and the system emptied and cleaned ready for a new culture. The nutrients and / or culture additives may be added at the beginning of culture or during the culture. Continuous culture refers to culturing cells in a system whereby cells are continuously removed after a period of growth, or removed at specific points in time, while a population of cells remain in the system which are able to continue to grow and divide. This process is repeated for a set period of time or indefinitely. The nutrients and / or culture additives are added periodically or continuously so that the cells present in the system always have optimum conditions in which to grow and divide.

[0214] In another embodiment the invention provides cultivated animal tissue comprising the modified cell according to any previous embodiments of the invention.

[0215] In another aspect, the invention provides the use of the modified animal cell according to any previous embodiments for cellular agriculture.

[0216] In another aspect, the invention provides a method for producing an immortalised cell line comprising a method according to any previous aspects of the invention, wherein the immortalised cell line comprises at least one of the aforementioned genetic modifications.

[0217] In a further aspect, the invention provides a method of producing a cultured meat product comprising culturing the one or more modified animal non-human cells or cell line according to any previous embodiments and optionally forming the cells into a tissue like structure. In a related embodiment, the method comprises forming the cells into a muscle tissue like structure. In a further aspect, the invention provides a cultured meat product for human or non-human consumption comprising a modified cell or cell line of the invention.

[0218] In a particular embodiment, a cultured meat product refers to a product in which cells according to the invention are formed into a product that is acceptable and / or suitable and / or appropriate for human consumption. The product may be of a structure that mimics or is intended to mimic the tissue of animal species which are used for human consumption. The cultured meat product 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 that is used for human consumption.

[0219] A tissue like structure according to the invention is a structure that resembles the specific tissue of an animal in terms of texture, taste, mouthfeel, visual structure, visual texture and colour. The tissue like structure does not have to be able to carry out the bodily functions that the tissue would carry out in vivo. Tissue like structure is intended to mean that the tissue like structure appears similar or the same as tissue taken from the animal to a consumer of the cultured meat product.

[0220] The cultured meat product comprises modified cells according to the invention but may additionally comprise other components such as colourant, flavourings and / or flavour enhancing compositions and dietary supplements such as vitamins and / or minerals.

[0221] Also provided is a packaged cultivated meat product comprising or derived from a cell or cell line of the invention. Genetically modified cell lines

[0222] A cell line for use in the aforementioned method is defined below. However, based on the teaching of the method and as defined in Applicant’s own PCT / GB2023 / 052528 and PCT / GB2023 / 053374, which are hereby incorporated by reference, the skilled person would be aware of other cell lines which may be used with the method of culturing an animal cell in reduced serum and growth factor media or serum and growth factor free media.

[0223] According to an aspect of the invention, there is provided a cultured animal cell having a genetic modification in the PTEN gene and / or SPRY gene and wherein the animal is of an animal species suitable for human or animal consumption. The advantage of the cell line of this aspect of the invention is that it reduced the doubling time. A reduction of the doubling time enhances the commercialisation and efficiency of cultured meat. Another advantage of the cell line is that it tolerates media with low concentrations of growth factors and / or absence of serum and / or absence of insulin betterthan cell lines without those genetic modifications. Growth in media without / with reduced concentrations of growth factors and serum reduces production costs massively and enhances consumer acceptance of the end product. Another advantage of those cells is that they show increased cell size, which can enhance economic viability of the process as the same number of cells would produce more product mass. These advantages may be measured in comparison to a control cell line which does not comprise a genetic modification.

[0224] In one embodiment, wherein the SPRY gene is selected from a group consisting of SPRY1 , SPRY2, SPRY3, and / or SPRY4. In one embodiment, the SPRY gene is SPRY2.

[0225] As described above, the animal used in various aspects of the invention may be of an animal species used in agriculture. An animal species used in is an animal farmed for human. Such animals are listed above. In a preferred embodiment, they include pig, bovine (e.g. cattle), poultry (e.g. chicken, turkey, duck, geese), sheep, goat, Equidae, Camelidae, fish, crustaceans or mollusc.

[0226] In one embodiment, the cultured animal cell is a sus scrofa wild type (Duroc) cell. The PTEN nucleic acid coding sequence for sus scrofa wild type (Duroc) is as shown in SEQ ID NO: 1 and the PTEN protein sequence for sus scrofa wild type (Duroc) is shown in SEQ ID NO: 2.

[0227] In one embodiment, the cultured animal cell is a sus scrofa Largewhite cell. The PTEN nucleic acid coding sequence for sus scrofa Largewhite is as shown in SEQ ID NO: 3 and the PTEN protein sequence for sus scrofa Largewhite is shown in SEQ ID NO: 4. In one embodiment, the cultured animal cell is a Bos taurus cell. The PTEN nucleic acid coding sequence for Bos taurus is as shown in SEQ ID NO: 5 and the PTEN protein sequence for Bos taurus is shown in SEQ ID NO: 6.

[0228] In one embodiment, the cultured animal cell is a Bos taurus cell. The SPRY1 nucleic acid coding sequence for Bos taurus is as shown in SEQ ID NO: 7 and the SPRY1 protein sequence for Bos taurus is shown in SEQ ID NO: 8.

[0229] In one embodiment, the cultured animal cell is a sus scrofa wild type (Duroc) cell. The SPRY2 nucleic acid coding sequence for sus scrofa wild type (Duroc) is as shown in SEQ ID NO: 9 and the SPRY2 protein sequence for sus scrofa wild type (Duroc) is shown in SEQ ID NO: 10.

[0230] In one embodiment, the cultured animal cell is a sus scrofa Largewhite cell. The SPRY2 nucleic acid coding sequence for sus scrofa Largewhite is as shown in SEQ ID NO: 11 and the SPRY2 protein sequence for sus scrofa Largewhite is shown in SEQ ID NO: 12.

[0231] In one embodiment, the cultured animal cell is a Bos taurus cell. The SPRY2 nucleic acid coding sequence for Bos taurus is as shown in SEQ ID NO: 13 and the SPRY2 protein sequence for Bos taurus is shown in SEQ ID NO: 14.

[0232] In one embodiment, the cultured animal cell is a Bos taurus cell. The SPRY3 nucleic acid coding sequence for Bos taurus is as shown in SEQ ID NO: 15 and the SPRY3 protein sequence for Bos taurus is shown in SEQ ID NO: 16.

[0233] In one embodiment, the cultured animal cell is a Bos taurus cell. The SPRY4 nucleic acid coding sequence for Bos taurus is as shown in SEQ ID NO: 15 and the SPRY4 protein sequence for Bos taurus is shown in SEQ ID NO: 16.

[0234] The animal cell may be a somatic cell and selected from one of the following cell types: myoblast, fibroblast, myofibroblast, adipose derived stem cell, epithelial cell, mesenchymal stem cell, satellite cell, iPSC, or hepatocyte.

[0235] Genetic modification in the PTEN gene and / or SPRY gene may be performed by any of the methods as set out above. For example, the expression of the PTEN gene and / or SPRY gene can be targeted in a number of different ways, including at least one of:

[0236] 1) gene level modification by: a. knock-out or reduced activity / transcription / translation levels via editing in coding sequences, promoters, introns, regulatory regions; b. RNA-directed DNA methylation; or c. transcription activation or repression using CRISPRa or CRISPRi or similar target specific methods; d. knock-out or reduced activity / transcription / translation levels via undirected means, for example radiation or chemical mutagenesis; e. overexpression of an endogenous nucleotide sequence

[0237] 2) post-transcription level (post-transcriptional gene silencing) modification by: a. RNAi or siRNA to reduce translation of mRNA into protein; or b. site specific nucleases to modify or cleave mRNA, such as CRISPR / Cas13a;

[0238] 3) post-translational level (protein disruption or activation) modification by: a. inclusion of activity blocking / reducing or enhancing molecules, wherein the activity blocking / reducing or enhancing molecules are small molecules, antibodies, or the like; or b. inclusion of protein degrading ingredients, wherein the protein degrading ingredients are specialised proteases, exoproteases, or endoproteases. c. Enhancing or reducing of protein activity through integration of activating or reducing functional mutations in the corresponding gene sequences.

[0239] In one embodiment, expression of the PTEN gene and / or SPRY genes may be modified by disrupting expression of a functional protein from the gene(s). In a related embodiment, CRISPR may be used to knockout the PTEN gene and / or SPRY genes. In a further embodiment, single point mutation or multiple mutations may be used to knock out the PTEN gene and / or SPRY gene expression. For example, the CRISPR / Cas9 or CRISPR / Cas13a systems may be used to knock out PTEN gene and / or SPRY gene expression. However, any of the techniques described above may be suitable for creating the genetic modification.

[0240] The genetic modification in the PTEN gene and / or SPRY gene may be used in combination with other genetic modifications in order to further assist in reducing the doubling time. For example, see Applicant’s own Patent Application PCT / GB2023 / 052528 and Patent Applications PCT / GB2023 / 053374, which are hereby incorporated by reference. Thus, in one embodiment, the at least one additional genetic modification may be in one or more of the following genes: RB1, TP53, NF2 BAK1, BAX, and / or a RAS gene.

[0241] The genetic modification to the RB1, TP53, NF2, BAK1, BAX, and / or a RAS genes may be made in combination with the genetic modification in the PTEN gene and / or SPRY gene individually or in any combination. In one embodiment, the genetic modification may be a single point mutation or multiple mutations to knock out gene expression, e.g. for the RB1, TP53, BAK1, BAX, and NF2 genes, or the genetic modification may be a single point mutation or multiple mutations to knock in gene expression, e.g. for the RAS gene.

[0242] The animal cell may have the genetic modification in the PTEN gene and / or SPRY gene in combination with any of the following additional genetic modifications. In one embodiment, the animal cell has an additional genetic modification in the RB1 gene. In one embodiment, the animal cell has an additional genetic modification in the TP53 gene. In one embodiment, the animal cell has an additional genetic modification in NF2 gene. In one embodiment, the animal cell has an additional genetic modification in a RAS gene. In one embodiment, the animal cell has an additional genetic modification in a BAK1 gene. In one embodiment, the animal cell has an additional genetic modification in a BAX gene. In one embodiment, the animal cell has an additional genetic modification in the RB1 gene and in the TP53 gene. In one embodiment, the animal cell has an additional genetic modification in the RB1 gene and in the TP53 gene. In one embodiment, the animal cell has an additional genetic modification in the RB1 gene and in the NF2 gene. In one embodiment, the animal cell has an additional genetic modification in the RB1 gene and in a RAS gene. In one embodiment, the animal cell has an additional genetic modification in the RB1 gene and in the BAK1 gene. In one embodiment, the animal cell has an additional genetic modification in the RB1 gene and in a BAX gene. In one embodiment, the animal cell has an additional genetic modification in the TP53 gene and in the NF2 gene. In one embodiment, the animal cell has an additional genetic modification in the TP53 gene and in a RAS gene. In one embodiment, the animal cell has an additional genetic modification in the TP53 gene and in the BAK1 gene. In one embodiment, the animal cell has an additional genetic modification in the TP53 gene and in a BAX gene. In one embodiment, the animal cell has an additional genetic modification in the NF2 gene and in a RAS gene. In one embodiment, the animal cell has an additional genetic modification in the NF2 gene and in the BAK1 gene. In one embodiment, the animal cell has an additional genetic modification in the NF2 gene and in a BAX gene. In one embodiment, the animal cell has an additional genetic modification in a RAS gene and in the BAK1 gene. In one embodiment, the animal cell has an additional genetic modification in a RAS gene and in a BAX gene. In one embodiment, the animal cell has an additional genetic modification in the BAK1 gene and in a BAX gene. In one embodiment, the animal cell has additional genetic modifications in the RB1 gene, the TP53 gene and in the NF2 gene. In one embodiment, the animal cell has additional genetic modifications in the RB1 gene, the TP53 gene and in a RAS gene. In one embodiment, the animal cell has additional genetic modifications in the RB1 gene, the TP53 gene and in the BAK1 gene. In one embodiment, the animal cell has additional genetic modifications in the RB1 gene, the TP53 gene and in a BAX gene. In one embodiment, the animal cell has additional genetic modifications in the RB1 gene, the NF2 gene and in a RAS gene. In one embodiment, the animal cell has additional genetic modifications in the RB1 gene, the NF2 gene and in a BAX' gene. In one embodiment, the animal cell has additional genetic modifications in the RB1 gene, a RAS gene and in a BAX' gene. In one embodiment, the animal cell has additional genetic modifications in the RB1 gene, the BAK1 gene and in a BAX'gene. In one embodiment, the animal cell has additional genetic modifications in the TP53 gene, the NF2 gene and in a RAS gene. In one embodiment, the animal cell has additional genetic modifications in the TP53 gene, the NF2 gene and in the BAK1 gene. In one embodiment, the animal cell has additional genetic modifications in the TP53 gene, the NF2 gene and in the BAK1 gene. In one embodiment, the animal cell has additional genetic modifications in the TP53 gene, the NF2 gene and in the BAK1 gene. In one embodiment, the animal cell has additional genetic modifications in the TP53 gene, the NF2 gene and in a BAX gene. In one embodiment, the animal cell has additional genetic modifications in the TP53 gene, a RAS gene and in the BAK1 gene. In one embodiment, the animal cell has additional genetic modifications in the TP53 gene, a RAS gene and in a BAX' gene. In one embodiment, the animal cell has additional genetic modifications in the TP53 gene, the BAK1 gene and in a BAX' gene. In one embodiment, the animal cell has additional genetic modifications in the NF2 gene, a RAS gene and in the BAK1 gene. In one embodiment, the animal cell has additional genetic modifications in the NF2 gene, a RAS gene and in the BAX' gene. In one embodiment, the animal cell has additional genetic modifications in the NF2 gene, the BAK1 gene and in the BAX' gene. In one embodiment, the animal cell has additional genetic modifications in a RAS gene, the BAK1 gene and in a BAX' gene. In one embodiment, the animal cell has additional genetic modifications in the RB1 gene, the TP53 gene, the NF2 gene, and in a RAS gene. In one embodiment, the animal cell has additional genetic modifications in the RB1 gene, the TP53 gene, the NF2 gene, and in the BAK1 gene. In one embodiment, the animal cell has additional genetic modifications in the RB1 gene, the TP53 gene, the NF2 gene, and in a BAX' gene. In one embodiment, the animal cell has additional genetic modifications in the RB1 gene, the TP53 gene, a RAS gene, and in a BAX' gene. In one embodiment, the animal cell has additional genetic modifications in the RB1 gene, the NF2 gene, a RAS gene, and in the BAK1 gene. In one embodiment, the animal cell has additional genetic modifications in the RB1 gene, the NF2 gene, the BAK1 gene, and in a BAX' gene. In one embodiment, the animal cell has additional genetic modifications in the RB1 gene, a RAS gene, the BAK1 gene, and in a BAX' gene. In one embodiment, the animal cell has additional genetic modifications in the TP53 gene, the NF2 gene, a RAS gene, and in the BAK1 gene. In one embodiment, the animal cell has additional genetic modifications in the TP53 gene, the NF2 gene, a RAS gene, and in a BAX' gene. In one embodiment, the animal cell has additional genetic modifications in the TP53 gene, the NF2 gene, the BAK1 gene, and in a BAX' gene. In one embodiment, the animal cell has additional genetic modifications in the NF2 gene, a RAS gene, the BAK1 gene, and in a BAX' gene. In one embodiment, the animal cell has additional genetic modifications in the RB1 gene, the TP53 gene, the NF2 gene, a RAS gene and in the BAK1 gene. In one embodiment, the animal cell has additional genetic modifications in the RB1 gene, the TP53 gene, the NF2 gene, a RAS gene and a BAX' gene. In one embodiment, the animal cell has additional genetic modifications in the RB1 gene, the TP53 gene, the NF2 gene, the BAK1 gene and a BAX' gene. In one embodiment, the animal cell has additional genetic modifications in the RB1 gene, the TP53 gene, a RAS gene, the BAK1 gene and a BAX' gene. In one embodiment, the animal cell has additional genetic modifications in the RB1 gene, the NF2 gene, a RAS gene, the BAK1 gene and a BAX' gene. In one embodiment, the animal cell has additional genetic modifications in the TP53 gene, the NF2 gene, a RAS gene, the BAK1 gene and a BAX' gene. Suitable sequence from genes in pig (Sus scrofa) are described as follows (see also table 1). Thus, the modified cell may be a pig cell and the targeted gene is selected from RB1, TP53, NF2, and / or HRAS, NRAS, or KRAS.

[0243] Further, examples for suitable genetic modifications to RAS genes in porcine and bovine species are set out in Applicant’s own PCT / GB2023 / 052528 and PCT / GB2023 / 053374, which are hereby incorporated by reference.

[0244] A skilled person would know that for the manipulation of other animal cells from animal species suitable for human or animal consumption, e.g. suitable for human consumption, e.g. suitable for animal consumption, e.g. used in agriculture, e.g. as listed herein, the equivalent orthologue, i.e. the endogenous RB1, TP53, NF2, and / or HRAS gene specific to the non-human animal species targeted is to be genetically modified. Suitable gene sequences can be identified from public databases. A skilled person would also be able to identify suitable sequences using standard methods in the art to identify homologs and orthologs, for example based on sequence identity with the pig sequences.

[0245] In a further embodiment of the invention, the gene is RAS and the modification is a hyperactivation modification. In one embodiment, the resulting modifications in the RAS protein keep it in a constantly active state. In one embodiment, activating modifications reduce GTP hydrolysis. The RAS gene may be selected from any one of HRAS, NRAS, or KRAS (isoform A or isoform B). In a related embodiment of the invention, the hyperactivation modification comprises one or more amino acid substitutions in the protein. The RAS gene may be a porcine RAS gene. The RAS gene may be a bovine RAS gene. In one embodiment, gain of function mutations are introduced into the RAS genes using targeted nucleases or derivations thereof. In another embodiment, gain of function versions of the RAS proteins are introduced as exogenous nucleotides into the cells. In one embodiment, the gain of function mutations are introduced via random mutagenesis, including but not limited to chemical mutagenesis. In one embodiment, the gain of function mutations are introduced by spontaneous mutations. In another embodiment, RAS activity is increased by perturbing other regulators of RAS activity. In another embodiment, RAS activity is increased by expressing the RAS gene via a non-native promoter.

[0246] For example, in one embodiment, the animal cell may be a sus scrofa var. Largewhite in which the HRAS gene comprises a hyperactivation modification and has a nucleic acid sequence according to SEQ ID NO: 26 and a protein sequence according to SEQ ID NO: 27.

[0247] In one embodiment, the animal cell may be a sus scrofa var. Largewhite in which the NRAS gene comprises a hyperactivation modification and has a nucleic acid sequence according to SEQ ID NO: 28 and a protein sequence according to SEQ ID NO: 29. In one embodiment, the animal cell may be a sus scrota var. Largewhite in which the KRAS gene comprises a hyperactivation modification and has a nucleic acid sequence according to SEQ ID NO: 30 and a protein sequence according to SEQ ID NO: 31 .

[0248] Further examples are described in detail in Applicant’s own PCT / GB2023 / 052528 and PCT / GB2023 / 053374, which are hereby incorporated by reference.

[0249] In one aspect of the invention, there is provided a method of producing cultivated meat or a cultured meat product comprising culturing an animal cell having a genetic modification in the PTEN gene and / or SPRY gene. In a further aspect of the invention, there is provided a method of producing a cultured animal cell having a genetic modification in the PTEN gene and / or SPRY gene. In a yet further aspect of the invention, there is provided a cultivated or cultured animal tissue or a cultivated or cultured meat product comprising a modified animal cell having a genetic modification in the PTEN gene and / or SPRY gene.

[0250] The invention also relates to use of a modified cultured animal cell having a genetic modification in the PTEN gene and / or SPRY gene for cellular agriculture.

[0251] In one aspect of the invention, there is provided a method of producing cultivated meat or a cultured meat product comprising culturing an animal cell expressing activated versions of one or more RAS proteins. In a further aspect of the invention, there is provided a method of producing a cultured animal cell expressing activated versions of one or more RAS proteins. In a yet further aspect of the invention, there is provided a cultivated or cultured animal tissue or a cultivated or cultured meat product comprising a modified animal cell expressing activated versions of one or more RAS proteins.

[0252] The invention also relates to use of a modified cultured animal cell expressing activated versions of one or more RAS proteins for cellular agriculture.

[0253] According to an aspect of the invention, there is provided a modified animal cell comprising a genetic modification in the BAK1 gene and / or the BAX gene, and wherein the animal is of an animal species suitable for human or animal consumption. The advantage of the cell line of this aspect of the invention is that deletion or suppression of the pro-apoptotic proteins BAX and BAK1 has been shown to confer apoptosis resistance, improving cell viability, increasing density, increasing cell size and increasing lifespan. These advantages are particularly relevant for cultivated meat production, as larger cells contribute more biomass per cell, improving overall yield and product quality. When scaling up to industrial bioreactors, even small increases in cell size can have a substantial impact on total biomass output. As described in examples 18-21 set out below, apoptotic cell death poses a significant challenge in large-scale cell culture systems, such as bioreactors used for cultivated meat production, as it leads to reduced productivity and lower overall yield. Apoptosis can be triggered by nutrient depletion at the end of batch culture, as well as stress factors like increased osmolality and shear stress. The apoptosis resistance, improved cell viability, increased cell density, increased cell size and / or increased cell lifespan of a cultivated or cultured animal cell suitable for human or animal consumption may be in comparison to a control cell line which does not comprise a genetic modification in the BAK1 gene and / or the BAX gene, as appropriate.

[0254] In one embodiment, modified animal cell comprises a genetic modification in the BAK1 gene and the BAX gene.

[0255] In one embodiment, there is provided a modified animal cell comprising a genetic modification in the BAK1 gene and / or the BAX gene, wherein the cells have apoptosis resistance, improving cell viability, density, and / or lifespan, and wherein the animal is of an animal species suitable for human or animal consumption.

[0256] As described above, the animal used in various aspects of the invention may be of an animal species used in agriculture. An animal species used in is an animal farmed for human. Such animals are listed above. In a preferred embodiment, they include pig, bovine (e.g. cattle), poultry (e.g. chicken, turkey, duck, geese), sheep, goat, Equidae, Camelidae, fish, crustaceans or mollusc. In one preferred embodiment, the animal cells are wagyu bovine cells.

[0257] In one embodiment, the animal cell is a somatic cell.

[0258] In one embodiment, the animal cell is selected from one of the following cell types: myoblast, fibroblast, myofibroblast, adipose derived stem cell, epithelial cell, mesenchymal stem cell, satellite cell, iPSC, or hepatocyte.

[0259] In one embodiment, the cultured animal cell is a Bos taurus cell. The BAX nucleic acid coding sequence for Bos taurus is as shown in SEQ ID NO: 37 and the BAX protein sequence for Bos taurus is shown in SEQ ID NO: 38.

[0260] In one embodiment, the cultured animal cell is a Bos taurus cell. The BAK1 nucleic acid coding sequence for Bos taurus is as shown in SEQ ID NO: 39 and the BAK1 protein sequence for Bos taurus is shown in SEQ ID NO:40.

[0261] Genetic modification in the BAK1 gene and / or the BAX gene may be performed by any of the methods as set out above. For example, the expression of the BAK1 gene and / or the BAX gene can be targeted in a number of different ways, including at least one of:

[0262] 1) gene level modification by: a. knock-out or reduced activity / transcription / translation levels via editing in coding sequences, promoters, introns, regulatory regions; b. RNA-directed DNA methylation; or c. transcription activation or repression using CRISPRa or CRISPRi or similar target specific methods; d. knock-out or reduced activity / transcription / translation levels via undirected means, for example radiation or chemical mutagenesis; e. overexpression of an endogenous nucleotide sequence

[0263] 2) post-transcription level (post-transcriptional gene silencing) modification by: a. RNAi or siRNA to reduce translation of mRNA into protein; or b. site specific nucleases to modify or cleave mRNA, such as CRISPR / Cas13a;

[0264] 3) post-translational level (protein disruption or activation) modification by: a. inclusion of activity blocking / reducing or enhancing molecules, wherein the activity blocking / reducing or enhancing molecules are small molecules, antibodies, or the like; or b. inclusion of protein degrading ingredients, wherein the protein degrading ingredients are specialised proteases, exoproteases, or endoproteases. c. Enhancing or reducing of protein activity through integration of activating or reducing functional mutations in the corresponding gene sequences.

[0265] In one embodiment, the genetic modification is a gene is a loss of function modification or leads to reduction in function.

[0266] In one embodiment, the genetic modification comprises a knock-out of the BAK1 gene and / or the BAX gene or loss of protein function.

[0267] In one embodiment, the modification is introduced using targeted genome modification or randomised mutagenesis or by spontaneous mutation.

[0268] In one embodiment, the modification is in the promoter region or coding region of one or more genes.

[0269] In one embodiment, the modification is introduced using targeted genome modification, optionally using a targeted endonuclease.

[0270] In one embodiment, the endonuclease is selected from meganuclease, TALEN, ZFN or CRISPR, optionally CRISPR / Cas9. In one embodiment, expression of the BAK1 gene and / or the BAX gene may be modified by disrupting expression of a functional protein from the gene(s). In a related embodiment, CRISPR may be used to knockout the BAK1 gene and / orthe BAX gene. In a further embodiment, single point mutation or multiple mutations may be used to knock out the BAK1 gene and / or the BAX gene expression. For example, the CRISPR / Cas9 or CRISPR / Cas13a systems may be used to knock out BAK1 gene and / or the BAX gene expression. However, any of the techniques described above may be suitable for creating the genetic modification.

[0271] The genetic modification in the BAK gene and / or the BAX gene gene may be used in combination with other genetic modifications to further assist in reducing the doubling time. For example, see Applicant’s own Patent Application PCT / GB2023 / 052528 and Patent Applications PCT / GB2023 / 053374, which are hereby incorporated by reference. Thus, in one embodiment, the at least one additional genetic modification may be in one or more of the following genes: RB1, TP53, NF2, RAS, PTEN and / or SPRY gene.

[0272] The genetic modification to the RB1, TP53, NF2, RAS, PTEN and / or SPRY gene may be made in combination with the genetic modification in the BAK gene and / or BAX gene individually or in any combination. In one embodiment, the genetic modification may be a single point mutation or multiple mutations to knock out gene expression, e.g. for the RB1, TP53, and NF2 genes, or the genetic modification may be a single point mutation or multiple mutations to knock in gene expression, e.g. for the RAS gene.

[0273] The animal cell may have the genetic modification in the BAK gene and / or BAX gene in combination with any of the following additional genetic modifications. In one embodiment, the animal cell has an additional genetic modification in the RB1 gene. In one embodiment, the animal cell has an additional genetic modification in the TP53 gene. In one embodiment, the animal cell has an additional genetic modification in NF2 gene. In one embodiment, the animal cell has an additional genetic modification in a RAS gene. In one embodiment, the animal cell has an additional genetic modification in a PTEN gene. In one embodiment, the animal cell has an additional genetic modification in a SPRY gene. In one embodiment, the animal cell has an additional genetic modification in the RB1 gene and in the TP53 gene. In one embodiment, the animal cell has an additional genetic modification in the RB1 gene and in the TP53 gene. In one embodiment, the animal cell has an additional genetic modification in the RB1 gene and in the NF2 gene. In one embodiment, the animal cell has an additional genetic modification in the RB1 gene and in a RAS gene. In one embodiment, the animal cell has an additional genetic modification in the RB1 gene and in the PTEN gene. In one embodiment, the animal cell has an additional genetic modification in the RB1 gene and in a SPRY gene. In one embodiment, the animal cell has an additional genetic modification in the TP53 gene and in the NF2 gene. In one embodiment, the animal cell has an additional genetic modification in the TP53 gene and in a RAS gene. In one embodiment, the animal cell has an additional genetic modification in the TP53 gene and in the PTEN gene. In one embodiment, the animal cell has an additional genetic modification in the TP53 gene and in a SPRY gene. In one embodiment, the animal cell has an additional genetic modification in the NF2 gene and in a RAS gene. In one embodiment, the animal cell has an additional genetic modification in the NF2 gene and in the PTEN gene. In one embodiment, the animal cell has an additional genetic modification in the NF2 gene and in a SPRY gene. In one embodiment, the animal cell has an additional genetic modification in a RAS gene and in the PTEN gene. In one embodiment, the animal cell has an additional genetic modification in a RAS gene and in a SPRY gene. In one embodiment, the animal cell has an additional genetic modification in the PTEN gene and in a SPRYgene. In one embodiment, the animal cell has additional genetic modifications in the RB1 gene, the TP53 gene and in the NF2 gene. In one embodiment, the animal cell has additional genetic modifications in the RB1 gene, the TP53 gene and in a RAS gene. In one embodiment, the animal cell has additional genetic modifications in the RB1 gene, the TP53 gene and in the PTEN gene. In one embodiment, the animal cell has additional genetic modifications in the RB1 gene, the TP53 gene and in a SPRYgene. In one embodiment, the animal cell has additional genetic modifications in the RB1 gene, the NF2 gene and in a RAS gene. In one embodiment, the animal cell has additional genetic modifications in the RB1 gene, the NF2 gene and in a SPRY gene. In one embodiment, the animal cell has additional genetic modifications in the RB1 gene, a RAS gene and in a SPRY gene. In one embodiment, the animal cell has additional genetic modifications in the RB1 gene, the PTEN gene and in a SPRY gene. In one embodiment, the animal cell has additional genetic modifications in the TP53 gene, the NF2 gene and in a RAS gene. In one embodiment, the animal cell has additional genetic modifications in the TP53 gene, the NF2 gene and in the PTEN gene. In one embodiment, the animal cell has additional genetic modifications in the TP53 gene, the NF2 gene and in the PTEN gene. In one embodiment, the animal cell has additional genetic modifications in the TP53 gene, the NF2 gene and in the PTEN gene. In one embodiment, the animal cell has additional genetic modifications in the TP53 gene, the NF2 gene and in a SPRY gene. In one embodiment, the animal cell has additional genetic modifications in the TP53 gene, a RAS gene and in the PTEN gene. In one embodiment, the animal cell has additional genetic modifications in the TP53 gene, a RAS gene and in a SPRY gene. In one embodiment, the animal cell has additional genetic modifications in the TP53 gene, the PTEN gene and in a SPRY gene. In one embodiment, the animal cell has additional genetic modifications in the NF2 gene, a RAS gene and in the PTEN gene. In one embodiment, the animal cell has additional genetic modifications in the NF2 gene, a RAS gene and in the SPRY gene. In one embodiment, the animal cell has additional genetic modifications in the NF2 gene, the PTEN gene and in the SPRYgene. In one embodiment, the animal cell has additional genetic modifications in a RXIS gene, the PTEN gene and in a SPRYgene. In one embodiment, the animal cell has additional genetic modifications in the RB1 gene, the TP53 gene, the NF2 gene, and in a RAS gene. In one embodiment, the animal cell has additional genetic modifications in the RB1 gene, the TP53 gene, the NF2 gene, and in the PTEN gene. In one embodiment, the animal cell has additional genetic modifications in the RB1 gene, the TP53 gene, the NF2 gene, and in a SPRY gene. In one embodiment, the animal cell has additional genetic modifications in the RB1 gene, the TP53 gene, a RAS gene, and in a SPRY gene. In one embodiment, the animal cell has additional genetic modifications in the RB1 gene, the NF2 gene, a RAS gene, and in the PTEN gene. In one embodiment, the animal cell has additional genetic modifications in the RB1 gene, the NF2 gene, the PTEN gene, and in a SPRY gene. In one embodiment, the animal cell has additional genetic modifications in the RB1 gene, a RAS gene, the PTEN gene, and in a SPRY gene. In one embodiment, the animal cell has additional genetic modifications in the TP53 gene, the A / F2 gene, a RAS gene, and in the PTEN gene. In one embodiment, the animal cell has additional genetic modifications in the TP53 gene, the NF2 gene, a RAS gene, and in a SPRY gene. In one embodiment, the animal cell has additional genetic modifications in the TP53 gene, the NF2 gene, the PTEN gene, and in a SPRY gene. In one embodiment, the animal cell has additional genetic modifications in the NF2 gene, a RAS gene, the PTEN gene, and in a SPRY gene. In one embodiment, the animal cell has additional genetic modifications in the RB1 gene, the TP53 gene, the NF2 gene, a RAS gene and in the PTEN gene. In one embodiment, the animal cell has additional genetic modifications in the RB1 gene, the TP53 gene, the NF2 gene, a RAS gene and a SPRY gene. In one embodiment, the animal cell has additional genetic modifications in the RB1 gene, the TP53 gene, the NF2 gene, the PTEN gene and a SPRY gene. In one embodiment, the animal cell has additional genetic modifications in the RB1 gene, the TP53 gene, a RAS gene, the PTEN gene and a SPRY gene. In one embodiment, the animal cell has additional genetic modifications in the RB1 gene, the NF2 gene, a RAS gene, the PTEN gene and a SPRY gene. In one embodiment, the animal cell has additional genetic modifications in the TP53 gene, the NF2 gene, a RAS gene, the PTEN gene and a SPRYgene.

[0274] Suitable sequence from genes in cow (Bos taurus) are described as follows (see also table 1). Thus, the modified cell may be a cow cell and the targeted gene is selected from RB1, TP53, NF2, HRAS, NRAS, KRAS, PTEN, SPRY1, SPRY2, SPRY3, and / or SPRY4.

[0275] Further, examples for suitable genetic modifications to RAS genes in porcine and bovine species are set out in Applicant’s own PCT / GB2023 / 052528 and PCT / GB2023 / 053374, which are hereby incorporated by reference.

[0276] A skilled person would know that for the manipulation of other animal cells from animal species suitable for human or animal consumption, e.g. suitable for human consumption, e.g. suitable for animal consumption, e.g. used in agriculture, e.g. as listed herein, the equivalent orthologue, i.e. the endogenous RB1, TP53, NF2, and / or HRAS gene specific to the non-human animal species targeted is to be genetically modified. Suitable gene sequences can be identified from public databases. A skilled person would also be able to identify suitable sequences using standard methods in the art to identify homologs and orthologs, for example based on sequence identity with the pig sequences.

[0277] According to a tenth aspect of the invention, there is provided a method of producing cultivated meat or a cultured meat product comprising culturing the modified animal cell according to the invention. According to an eleventh aspect of the invention, there is provided a method of conferring apoptosis resistance, improving cell viability, increasing cell density, increasing cell size and / or increasing cell lifespan of a cultivated or cultured animal cell suitable for human or animal consumption comprising cultivating or culturing animal cells comprising a genetic modification in the BAK1 gene and / or the BAX gene. In one embodiment the method comprises cultivating or culturing animal cells comprising a genetic modification in the BAK1 gene and the BAX gene. The apoptosis resistance, improved cell viability, increased cell density, increased cell size and / or increased cell lifespan of a cultivated or cultured animal cell suitable for human or animal consumption may be in comparison to a control cell line which does not comprise a genetic modification in the BAK1 gene and / or the BAX gene, as appropriate.

[0278] In one embodiment, the modification reduces apoptosis, increases viability, increases density, increases cell size and / or increases lifespan 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%, or 95% in comparison to the unmodified animal cell, or about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40- fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold or 100-fold in comparison to the unmodified animal cell.

[0279] According to an twelfth aspect of the invention, there is provided a use of the modified cultured animal cell according to the invention for cellular agriculture.

[0280] According to a thirteenth aspect of the invention, there is provided a guide RNA targeting the sequence for the BAK1 gene or the sequence for the BAX' gene in a cultured animal cell according to any of claims 55 to 66.

[0281] In a further aspect of the invention, there is provided a method of culturing an animal cell comprising culturing the animal cell in a cell culture medium, wherein the cell culture medium is serum, exogenous growth factor, and / or insulin free or wherein the cell culture medium is reduced in serum, at least one exogenous growth factors, and / or insulin, and wherein the animal cell comprises a genetic modification selected from Table 1.

[0282] In a further aspect of the invention, there is provided a modified animal cell comprising a genetic modification selected from Table 1 , and wherein the animal is of an animal species suitable for human or animal consumption.

[0283] Guide RNA and kits

[0284] In another aspect, the invention provides a guide RNA targeting the sequence for the PTEN gene or the sequences for any of the SPRY genes in a cultured animal cell as described herein. In another aspect, the invention provides a guide RNA targeting the sequence for the BAK1 gene or the sequence for the BAX' gene in a cultured animal cell according to the invention.

[0285] In a further embodiment the invention provides a guide RNA according to any previous embodiment of the invention for use in a method of producing a modified cell according to any previous embodiment of the invention. In a related embodiment the invention provides a guide RNA according to the previous embodiment of the invention wherein the modified cell is a modified cell according to any previous embodiment of the invention.

[0286] In a further embodiment the invention provides a kit of parts comprising at least one of the guide RNA according as described above. In one embodiment, the guide RNA may be a chemically synthesized sgRNA. In a related embodiment, the chemically synthesized sgRNA may be used to perform CRISPR in conjunction with a Cas9 recombinant purified protein.

[0287] As explained above, in some embodiments, the methods of the invention use gene editing using sequence specific endonucleases that target one or more genes in an animal cell of interest. As also explained, Cas9 and gRNA may be comprised in a single or two expression vectors. The sgRNA targets the one or more gene nucleic acid sequence.

[0288] Thus, 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 the one or more genes.

[0289] In one embodiment, the guide RNA targets the sequence for the PTEN gene and comprises a sequence according to SEQ ID NO: 19 (porcine) or SEQ ID NO: 20 (bovine).

[0290] In one embodiment, the guide RNA targets the sequence for the SPRY1 gene and comprises a sequence according to SEQ ID NO: 21 (bovine).

[0291] In one embodiment, the guide RNA targets the sequence for the SPRY2 gene and comprises sequence according to SEQ ID NO: 22 (porcine) or SEQ ID NO: 23 (bovine).

[0292] In one embodiment, the guide RNA targets the sequence for the SPRY3 gene and comprises a sequence according to SEQ ID NO: 24 (bovine).

[0293] In one embodiment, the guide RNA targets the sequence for the SPRY4 gene and comprises a sequence according to SEQ ID NO: 25 (bovine).

[0294] In one embodiment, the guide RNA targets the sequence for the BAK1 gene and comprises a sequence according to SEQ ID NO: 42. In one embodiment, the guide RNA targets the sequence for the BAX gene and comprises a sequence according to SEQ ID NO: 41 .

[0295] In one embodiment, the nucleic acid sequence encodes at least one protospacer element.

[0296] In one embodiment, the construct further comprises a nucleic acid sequence encoding a CRISPR RNA (crRNA) sequence, wherein said crRNA sequence comprises the protospacer element sequence and additional nucleotides. In one embodiment, the construct further comprises a nucleic acid sequence encoding a transactivating RNA (tracrRNA).

[0297] In a further embodiment, the construct encodes at least one single-guide RNA (sgRNA), wherein said sgRNA comprises the tracrRNA sequence and the crRNA sequence. The sgRNA can be used for manipulation of animal cells. In another aspect of the invention, there is provided a nucleic acid construct comprising a DNA donor nucleic acid wherein said DNA donor nucleic acid is operably linked to a regulatory sequence. The regulatory sequence may be one or more of the following: intron, promoter and / or terminator.

[0298] Cas9 and sgRNA may be combined or in separate expression vectors (or nucleic acid constructs, such terms are used interchangeably). Similarly, Cas9, sgRNA and the donor DNA sequence may be combined or in separate expression vectors. In other words, in one embodiment, an isolated animal cell is transfected with a single nucleic acid construct comprising both sgRNA and Cas9 or sgRNA, Cas9 and the donor DNA sequence as described in detail above. In an alternative embodiment, an isolated animal cell is 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 the donor DNA sequence as defined above. The second and / or third nucleic acid construct may be transfected before, after or concurrently with the first and / or second nucleic acid construct. The advantage of a separate, second construct comprising a Cas protein is that the nucleic acid construct encoding at least one sgRNA can be paired with any type of Cas protein, as described herein, and therefore is not limited to a single Cas function (as would be the case when both Cas and sgRNA are encoded on the same nucleic acid construct).

[0299] In one embodiment, a construct as described above is operably linked to a promoter, for example a constitutive promoter.

[0300] 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. 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 DNA-cleavage domain thereof. More preferably, the endonuclease is Fokl.

[0301] In another aspect of the invention there is provided a single guide (sg) RNA molecule wherein said sgRNA comprises a crRNA sequence and a tracrRNA sequence. In one embodiment, the sgRNA molecule may comprise at least one chemical modification, for example that enhances its stability and / or binding affinity to the target sequence or the crRNA sequence to the tracrRNA sequence. For example, the crRNA may comprise a phosphorothioate backbone modification, such as "-fluoro (”-F), "-0-methyl ("-0-Me) and S-constrained ethyl (cET) substitutions.

[0302] In a further embodiment, the nucleic acid construct may further comprise at least one nucleic acid sequence encoding an endoribonuclease cleavage site. Preferably the endoribonuclease is Csy4 (also known as Cas6f). Where the nucleic acid construct comprises multiple sgRNA nucleic acid sequences the construct may comprise the same number of endoribonuclease cleavage sites. Accordingly, each sgRNA nucleic acid sequence is flanked by an endoribonuclease cleavage site. The term "variant" refers to a nucleotide sequence where the nucleotides are substantially identical to one of the above sequences. The variant may be achieved by modifications such as insertion, substitution or deletion of 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 described sequences. In one embodiment, sequence identity is at least 90%. In another embodiment, sequence identity is 100%. Sequence identity can be determined by any one known sequence alignment program in the art.

[0303] The invention also relates to a nucleic acid construct comprising a nucleic acid sequence operably linked to a suitable animal promoter. A suitable animal promoter may be a constitutive or strong promoter or may be a tissue-specific promoter. In one embodiment, suitable animal promoters are selected from, but not limited to, PGK, CMV, EF1 a, CAG, SV40 and Ubc.

[0304] The nucleic acid construct of the present invention may also further comprise a nucleic acid sequence that encodes a CRISPR enzyme. In a specific embodiment Cas9 is codon-optimised Cas9. In another embodiment, the CRISPR enzyme is a protein from the family of Class 2 candidate proteins, such as C2c1 , C2C2 and / or C2c3. In one embodiment, the Cas protein is from Streptococcus pyogenes. In an alternative embodiment, the Cas protein may be from any one of Staphylococcus aureus, Neisseria meningitides or Streptococcus thermophiles. The term "functional variant" as used herein with reference to Cas9 refers to a variant Cas9 gene sequence or part of the gene sequence which retains the biological function of the full non-variant sequence, for example, acts as a DNA endonuclease, or recognition or / and binding to DNA. A functional variant also comprises a variant of the gene of interest which has sequence alterations that do not affect function, for example non-conserved residues. Also encompassed is a variant that is substantially identical, i.e. has only some sequence variations, for example in non-conserved residues, compared to the wild type sequences as shown herein and is biologically active.

[0305] In a further embodiment, the Cas9 protein has been modified to improve activity. For example, in one embodiment, the Cas9 protein may comprise the D10A amino acid substitution, this nickase cleaves only the DNA strand that is complementary to and recognized by the gRNA. In an alternative embodiment, the Cas9 protein may alternatively or additionally comprise the H840A amino acid substitution, this nickase cleaves only the DNA strand that does not interact with the sRNA. In this embodiment, Cas9 may be used with a pair (i.e. two) sgRNA molecules (or a construct expressing such a pair) and as a result can cleave the target region on the opposite DNA strand, with the possibility of improving specificity by 100-1500 fold. In a further embodiment, the Cas9 protein may comprise a D1135E substitution. The Cas 9 protein may also be the VQR variant. Alternatively, the Cas protein may comprise a mutation in both nuclease domains, HNH and RuvC-like and therefore is catalytically inactive. Rather than cleaving the target strand, this catalytically inactive Cas protein can be used to prevent the transcription elongation process, leading to a loss of function of incompletely translated proteins when co-expressed with a sgRNA molecule. An example of a catalytically inactive protein is dead Cas9 (dCas9) caused by a point mutation in RuvC and / or the HNH nuclease domains.

[0306] In a further embodiment, a Cas protein, such as Cas9 may be further fused with a repression effector, such as a histone-modifying / DNA methylation enzyme or a Cytidine deaminase to effect site-directed mutagenesis. In the latter, the cytidine deaminase enzyme does not induce dsDNA breaks, but mediates the conversion of cytidine to uridine, thereby effecting a C to T (or G to A) substitution.

[0307] Suitable methods for producing the CRISPR nucleic acids and vectors system are known, and for example are published in Ran et al 2013, Nat Protoc 8, 2281-2308 (2013).

[0308] 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, the isolated animal cell is transfected with at least one nucleic acid construct as described herein and a second nucleic acid construct, wherein said second nucleic acid construct comprises a nucleic acid sequence encoding a Cas protein, preferably a Cas9 protein or a functional variant thereof. Preferably, the second nucleic acid construct is transfected before, after or concurrently with the first nucleic acid construct described herein. In an alternative aspect of the invention, the nucleic acid construct comprises at least one nucleic acid sequence that encodes a TAL effector. Targeted nucleases, e.g. Meganucleases, ZNF, TALEN, CRISPR nucleases and derivations thereof, such as (but not exclusively) PRIME editors, base editors, CRISPRi, amongst others may form part of the invention.

[0309] Also included in the scope of the invention, is the use of the nucleic acid constructs (CRISPR constructs) described above or the sgRNA molecules in any of the above described methods. For example, there is provided the use of the above CRISPR constructs or sgRNA molecules to modulate the activity of one or more gene as described herein. In particular, as described herein, the CRISPR constructs may be used to create loss of function or hyperactivation alleles.

[0310] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are 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 the present disclosure, including methods, as well as the best mode thereof, of making and using this disclosure, the following examples are provided to further enable those skilled in the art to practice this disclosure. However, those skilled in the art will appreciate that the specifics of these examples should not be read as limiting on the invention, the scope of which should be apprehended from the claims and equivalents thereof appended to this disclosure. Various further aspects and embodiments of the present disclosure will be apparent to those skilled in the art in view of the present disclosure.

[0311] All documents mentioned in this specification are incorporated herein by reference in their entirety, including references to gene accession numbers, scientific publications and references to patent publications.

[0312] The term “and / or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, “A and / or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

[0313] The optional features set out herein may be used either individually or in combination with each other where appropriate and particularly in the combinations as set out in the accompanying claims. The optional features for each aspect or exemplary embodiment of the invention as set out herein are also to be read as applicable to any other aspect or exemplary embodiments of the invention, where appropriate. In other words, the skilled person reading this specification should consider the optional features for each exemplary embodiment of the invention as interchangeable and combinable between different exemplary embodiments.

[0314] Attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference. All of the features disclosed in this specification (including any accompanying claims, and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0315] Each feature disclosed in this specification (including any accompanying claims, and drawings) may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

[0316] The invention is not restricted to the details of the foregoing embodiment(s). The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, and drawings), orto any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0317] The invention is further illustrated in the following non-limiting examples.

[0318] Example 1 : Enrichment of PTEN and SPRY2 knockout mutants in a pooled cell competition assay in serum free suspension cell culture media lacking growth factors

[0319] PTEN (Phosphatase and tensin homolog) is a phosphatase and tumour suppressor gene in humans, where it is involved in cell cycle regulation and preventing cells from dividing too rapidly. Its main function is to inhibit the PI3K / AKT / mTOR signalling pathway through dephosphorylation of PI3K. The PI3K / AKT / mTOR pathway promotes proliferation and growth and is activated by growth factors and insulin (amongst others).

[0320] SPRY2 (Sprouty 2) is a negative feedback regulator of multiple receptor tyrosine kinase pathways, which are cell receptors for growth factors and hormones. If those cell receptors are triggered by growth factors, they activate downstream effectors and trigger the Ras-Raf-MEK-ERK pathway (or MAPK / ERK pathway) which promotes cell proliferation (amongst others). SPRY2 is part of the SPRY gene family which also includes SPRY1 / 3 / 4 which are described to also be involved in negative response to receptor tyrosine kinase pathways.

[0321] These two genes were identified amongst multiple other genes due to their known role of negatively regulating growth factor response in cells as potentially beneficial targets in CRISPR knockout approaches to improve growth in low / no growth factor conditions. A competition assay was used to identify potential candidates forgenetic modification to provide reduced doubling time, in which we pooled cell lines containing individual mutations and subjected this mixed cell line population to different growth conditions with reduced growth factors. A multiplex PCR allowed us to follow depletion or enrichment of individual mutant cell lines over time in those conditions and therefore to identify the gene knockouts with the strongest growth improvement in reduced growth factor conditions.

[0322] Methods:

[0323] Porcine var. Largewhite CRISPR immortalised suspension cells (P53 / _, RBT / _, HRASG12VA, NF2Z) were edited in separate editing reactions targeting single growth factor pathway candidate genes, including PTEN (Seq ID No. 1 , Seq ID No. 2, Seq ID No. 3, Seq ID No. 4) and SPRY2, using one sgRNA per gene (Seq ID No. 19, Seq ID No. 22). Cells were edited using nucleofection with synthetic single guide RNAs and Strep. Pyogenes Cas9 protein and expanded in serum free suspension media with 105 ng / mL FGF, 175 ng / mL IGF1 , 20 ng / mL PDGF0, 0.7 ng / mL HGF. After expansion, equal numbers of cells containing individual gene mutations were pooled together and seeded in duplicate Erlenmeyer flasks containing serum free suspension media with no growth factors or with 105 ng / mL FGF, 20 ng / mL PDGF, 0.7 ng / mL HGF or with 175 ng / mL IGF, 20 ng / mL PDGF, 0.7 ng / mL HGF respectively. Cells were passaged every 3-4 days for a total of 9 growth periods (GP). At the end of every other growth period, genomic DNA was extracted, and the mutated genes of interest were amplified by a multiplexed PCR and subjected to next-generation sequencing to quantify the editing abundance of each gene within the cell pool. Data from next-generation sequencing was used to calculate the fold change over time in the percentage of edited sequencing reads relative to day 0 (time of pooling). A schematic of the procedure can be found in (a).

[0324] Results:

[0325] Enrichment and depletion data of specific mutants in pooled experiment from duplicate Erlenmeyer flasks (n1 / n2) cultured in serum free suspension media containing no growth factors (b), serum free suspension media without IGF1 (c) and serum free suspension media without FGF2 (d). The fold change is shown on a Iog10 scale, where enrichment is represented by a darker shade of grey and white represents a depletion in functional gene knockout or knock-in within the cell pool relative to dO. Note that condition (b) was ended after passage 2 due to lack of cell growth, and flask n2 in condition (c) was grown for 7 passages only. PTEN mutants show strong enrichment in all conditions, SPRY2 mutants show enrichment under certain conditions.

[0326] Example 2: The PTEN and SPRY2 genes can be edited efficiently in different cell types and species and the edits are retained in mixed cell populations cultured in media containing reduced growth factors.

[0327] Methods: Cells were edited using nucleofection with synthetic single guide RNAs (Seq ID No. 19 , 20, 21 , 22, 23, 24, 25) and Strep. Pyogenes Cas9 protein. Note that the same guide RNAs were used between varieties of a given species (e.g. bovine var. Wagyu and var. Angus). Target genes were PTEN, SPRY1 , SPRY2, SPRY3 and / or SPRY4 (Seq ID No. 1-18) Editing efficiencies in cell pools were verified at 2-3 time points post editing and / or post suspension growth assays in different conditions to screen for enrichment or depletion of desired mutations. Enrichment of a mutation of interest suggest a positive impact of a given mutation on cell growth, while depletion suggests a detrimental effect on cell health or growth. Editing efficiencies were measured by PCR amplification of target region, Sanger Sequencing and Synthego ICE analysis of the Sanger Sequencing file Edits were considered a knockout (KO) if they contain a frameshift mutation or indels of 21 + base pairs, Other Indels refers to percentage of sequences that contain an indel which does not lead to a frameshift, WT refers to percentage of sequences that are wild type, unknown refers to the percentage of Sanger sequencing reads that cannot be explained by the ICE analysis tool.

[0328] Note that in some cases, cells were co-edited with sgRNAs against the P53, RB1 , RAS genes to immortalize the cell lines (see Patent Application PCT / GB2023 / 052528, which is hereby incorporated by reference) and the NF2 gene (see Patent Application P44661 GB1) to reduce cell doubling time in suspension. In all other cases, cell lines already had those edits.

[0329] Results:

[0330] Editing data from (a) edited porcine myoblast cell pools from different timepoints for (i) PTEN knockout cell pools (ii) SPRY2 knockout cell pools (iii) PTEN / SPRY2 double knockout cell pools; (b) Bovine var. Angus ADSCs from different timepoints for (i) PTEN KO (ii) SPRY2 KO (iii) PTEN / SPRY2 KO and PTEN / SPRY1-4 KO (iv) cell pools; (c) Bovine var. Wagyu myoblasts from different timepoints with a PTEN / SPRY2 KO.

[0331] Discussion: Figure 2 (and Figure 16a, see also example 16 below for further details of Figure 16a) focuses on CRISPR editing efficiencies in PTEN and SPRY genes in cell pools. When editing a pool of cells, each cell can obtain a different outcome of the editing process in their target genes, ranging from a functional knockout mutation (KO), or a mutation with low likelihood of functional effect (e.g. a deletion of 3 bp = 1 amino acid only), which is described as “Other InDeis” in the figure, or no mutation at all (wildtype / WT). We use the ICE online tool to determine the frequencies of each event in a cell pool based on Sanger Sequencing reads, and include an “unknown” value which corresponds to portions of the sequencing reads which the tool cannot assign to any of the other categories.

[0332] We show in this example that we achieve high knockout efficiencies in cell pools across species / cell lines shortly after editing. We also show that we can do multiplex editing, meaning that cell lines can simultaneously be edited in multiple genes (e.g. PTEN / SPRY1-4 knockout). Note from Figure 2 the high knockout efficiencies specifically in PTEN, SPRY1 , SPRY2, and SPRY4 which stay stable or increase in the knockout pools over time in the tested conditions. This suggests a beneficial effects of those mutations on cell growth. Note that all Insertion or deletion mutations (InDei) that lead to a frameshift or are >21 bp in size were considered as Knockout mutations. As we have a cell pool with mixed populations of cells regarding their mutation status in the given target genes (knockout, wildtype, other indels), those cells might grow differently well in a given medium over time and e.g. cells with a functional KO could outcompete cells without mutations or the other way around. This will lead to changes in the % values of KO / WT / Other Indels when doing the Sanger Sequencing / ICE analysis at different time points of the experiment, e.g. during cell expansion after editing (see data from different time points post editing) or post functional growth assays (see “after x passages in x media” bars). If KO efficiencies of a given gene stay high or increase over time, it hints as a beneficial effect of that mutation compared to cells without a functional KO. This can be seen e.g. for PTEN / SPRY2 but also to some degree for SPRY1 / 3 / 4 post in Figures 2 and 16a.

[0333] In summary, this example proves our technology in general (efficient CRISPR editing) and also hints at a functional effect of that KO due to stable / increasing KO populations within our cell pools over time.

[0334] Example 3: The PTEN and SPRY2 knockouts provide a growth advantage in suspension culture in porcine myoblast cells

[0335] Methods: For suspension growth assays, cells were seeded using a seeding density of 100,000 cells / mL into Erlenmeyer flasks and grown at 150 rpm in suspension media supplemented with 2.5% FBS and varying concentrations of growth factors as indicated. Cells were cultured for at least 5 passages as indicated. Doubling times were compared to a PTEN<+ / +) / SPRY2<+ / +) control line (Ctrl).

[0336] Results: (a) Doubling times (i) and cell densities (ii) of porcine myoblast cell pools over 11 passages in suspension media supplemented with 2.5% FBS, 5.25 ng / mL FGF, 8.75 ng / mL IGF1 , 1 ng / mL PDGF0 and 0.035 ng / mL HGF. (b) Doubling times (i) and cell densities (ii) of porcine myoblast cell pools over 10 passages in suspension media supplemented with 2.5% FBS, 1 .05 ng / mL FGF, 8.75 ng / mL IGF1 , 1 ng / mL PDGF0 and 0.035 ng / mL HGF. (c) Doubling times (i) and cell densities (ii) of porcine myoblast cell pools over 5 passages in suspension media supplemented with 2.5% FBS, 1.05 ng / mL FGF, 1.75 ng / mL IGF1 , 0.2 ng / mL PDGF0 and 0.007 ng / mL HGF. (d) Combined doubling time data from (a). Average doubling time forthe Ctrl (PTEN <+ / +> / SPRY2 <+ / +>) is 26.9 h; 24.7 h PTEN cells; 24.2 h SPRY2( / )cells; 24.5 h PTEN( / ) / SPRY2( / )cells, (e) Combined doubling time data from (b). Average doubling time for the Ctrl (PTEN <+ / +SPRY2 <+ / +>) is 34.1 h; 26.3 h cells; 29 h SPRY2 cells; 26.5 h PTEN( / ) / SPRY2( / )cells, (f) Combined doubling time data from (c). Average doubling time for the Ctrl (PTEN(+ / +) / SPRY2 <+ / +>) is 34 h; 26.1 h cells; 28 h cells; 24.5 h PTEN < / ) / SPRY2 KO cells, (g) Doubling times (i) and cell densities (ii) of porcine myoblast cell pools over 5 passages in suspension media with 2.5% FBS and no additional growth factors followed by 5 passages in suspension media supplemented with 2.5% FBS and 1.05 ng / mL FGF2, as depicted by the dashed vertical line. Due to insufficient cell number, the Ctrl cells were not seeded beyond passage 3. Note that negative doubling times are not plotted, (h) Combined doubling time data from cells growing in suspension media supplemented with 2.5% FBS and 1.05 ng / mL FGF2 in (g). Average doubling time for the PTEN cells is 33.4 h and 29.9 h PTEN < / ) / SPRY2 cells. Discussion: Figures 3 / 4 / 6 / 16 (see also examples 4, 6, and 16 below for further details of Figures 4, 6, and 16) compare doubling times / viable cell densities / cell size of mutant cell pools from different species compared to control cell lines in different media conditions across passaging experiments. We show here that lines with a PTEN, SPRY2, PTEN / SPRY2, PTEN / SPRY1-4 knockout have reduced doubling times and higher viable cell densities compared to the corresponding control, which become even more obvious when growth factor concentrations are heavily reduced (1-1.05 ng / mL FGF2 only) or even removed completely (shown in bovine var. Angus only). We can also show in Figures 4 / 6 / 16 that the PTEN and PTEN / SPRY2 knockout cell lines lead to increased cell size across multiple cow varieties and cell types (myoblasts, ADSC). Cell size is an important characteristic for the cultivated meat market as the same number of cells will generate different weight of product depending on cell size.

[0337] Example 4: The PTEN and SPRY1 / 2 / 3 / 4 knockouts provide a growth advantage and cell size increase in suspension media without growth factors in bovine var. Angus ADSC.

[0338] Methods: For suspension growth assays, cells were seeded using a seeding density of 100,000 cells / mL into Erlenmeyer flasks and grown at 150 rpm in suspension media supplemented with or without 2.5% FBS and with or without 1 .05 ng / mL FGF2 as indicated. Cells were cultured for at least 4 passages. Doubling times and cell diameters were compared to a non-edited control line (Ctrl).

[0339] Results: (a) Doubling times (i) and cell densities (ii) of bovine ADSC Ctrl and PTENf7) pools over 10 passages in suspension media containing 2.5% FBS and 1.05 ng / mL FGF2. (b) Doubling times (i) and cell densities (ii) of bovine ADSC Ctrl, PTENf7), SPRY2< / ) and PTEN(- / -) / SPRY1 (- / -) / SPRY2(- / -) / SPRY3<- / -) / SPRY4( / )pools over 7 passages in suspension media containing 2.5% FBS and no growth factors, (c) Doubling times (i) and cell densities (ii) of bovine ADSC Ctrl, PTEN( / ), SPRY2( / )and PTEN( / ) / SPRY1(- / ) / SPRY2( / ) / SPRY3( / ) / SPRY4( / )pools over 4 passages in serum free suspension media without the addition of growth factors, (d) Combined doubling time data from (a). Average doubling time for the Ctrl (PTEN <+ / +SPRY1-4 <+ / +>) is 27.2 h and 26.2 h for PTEN cells, (e) Combined doubling time data from (b). Average doubling time for the Ctrl (PTEN <+ / +VSPRY1-4 <+ / +>) is 75.5 h; 37.5 h cells; 37.7 h PTEN <- / - SPRY2 cells; 35 h PTEN < / ) / SPRY1-4 w cells, (f) Combined cell size of bovine ADSC cell pools from the culture conditions in (a). Average cell size for the Ctrl (PTEN <+ / +> / SPRY1-4 <+ / +>) is 14.3 pm and 15 pm for PTEN( / )cells (g) Combined cell size of bovine ADSC cell pools from the culture conditions in (b). Average cell size for the Ctrl (PTEN(+ / +) / SPRY1-4(+ / +)) is 14.6 pm; 15.3 pm PTEN( / )cells; 15.3 pm PTEN < / ) / SPRY2 cells; 15.2 pm PTEN w / SPRYI -4 cells, (h) Combined cell size of bovine ADSC cell pools from the culture conditions in (c). Average cell size for the Ctrl (PTEN cells; 14.8 pm PTEN < / ) / SPRY2 w cells; 15 pm PTEN <-

[0340] Example 5: Bovine var. Angus ADSC containing a PTEN / SPRY2 double knockout reach a higher maximum viable cell density in a suspension culture overgrowth study. Methods: For suspension overgrowth studies, cells were seeded into triplicate Erlenmeyer flasks at 1 e5 cells / mL seeding density and grown in suspension media supplemented with 2.5% FBS and 1 .05 ng / mL FGF2 for 7 days (168 hrs). The growth of PTEN <- / ) / SPRY2( / )cells were compared to a PTEN (+ / +) / SPRY2 <+ / +> control line (Ctrl). Cell counts were recorded every 24 hrs from day 3-7 to calculate (i) viable cell density (ii) cell diameter and (iii) cell viability.

[0341] Results: The PTEN( / ) / SPRY2( / )cell line reaches higher viable cell densities than the PTEN(+ / +) / SPRY2(+ / +while maintaining similar viability.

[0342] Discussion: Figures 5 and 7 (see example 7 for further details regarding Figure 7) show overgrowth studies of PTEN / SPRY2 knockout cell lines in different cow species. Here, we show that the mutant cell pools reach higher cell density peaks than control lines without PTEN / SPRY2 knockouts in different media conditions.

[0343] Example 6: Bovine var. Wagyu myoblast cells containing a PTEN / SPRY2 double knockout have a lower doubling time in suspension culture conditions, a larger cell diameter, and grow well in low growth factor conditions.

[0344] Methods: For suspension growth assays, cells were seeded using a seeding density of 100,000 cells / mL into Erlenmeyer flasks and grown at 150 rpm in suspension media supplemented with 2.5% FBS and with growth factors as indicated. Cells were cultured for at least 4 passages. Doubling times (i), viable cell densities (ii), pooled doubling times (iii) and cell diameters (iv) were compared to a PTEN<+ / +) / SPRY2<+ / +) control line.

[0345] Results: (a) Growth characteristics of Bovine var. Wagyu myoblast cells containing a PTEN / SPRY2 double knockout grown in suspension media with 2.5% FBS, 5 ng / mL FGF, 1 ng / mL PDGF0, 10 ng / mL IGF1 and 0.1 ng / mL HGF. Average doubling time Ctrl line: 41.8 h, average doubling time PTEN( / _) / SPRY2( / ): 33.3 h, average diameter Ctrl line: 12.9 pm, average diameter PTEN( / ) / SPRY2( / ): 14.2 pm. (b) Growth characteristics of Bovine var. Wagyu myoblast cells containing a PTEN / SPRY2 double knockout grown in suspension media with 2.5% FBS and 1 ng / mL FGF only. Average doubling time Ctrl line: 67.5 h, average doubling time PTEN( / ) / SPRY2( / ): 38.2 h, average diameter Ctrl line: 13.1 pm, average diameter PTEN( / -) / SPRY2( / ): 14.1 pm.

[0346] Example 7: Bovine var. Wagyu myoblast cells containing a PTEN / SPRY2 double knockout reach a higher maximum viable cell density in suspension culture

[0347] Methods: For suspension overgrowth studies, cells were seeded into triplicate Erlenmeyer flasks at 1 e5 cells / mL seeding density and grown in suspension media supplemented with 2.5% FBS and growth factors as indicated for 7 days. The growth of PTEN < / ) / SPRY2( / )cells were compared to a PTEN (+ / +) / SPRY2 <+ / +> control line (Ctrl). Cell counts were recorded every 24 hrs from day 3-7 to calculate (i) viable cell density (ii) cell diameter and (iii) cell viability.

[0348] Results: (a) Growth characteristics of Bovine var. Wagyu myoblast cells grown in suspension media with 2.5% FBS, 5 ng / mL FGF, 1 ng / mL PDGFp, 10 ng / mL IGF1 and 0.1 ng / mL HGF. (b) Growth characteristics of Bovine var. Wagyu myoblast cells grown in suspension media with 2.5% FBS and 1 ng / mL FGF only. Note that the PTEN( / ) / SPRY2( / )cells reach higher peak density and have a larger diameter while maintaining viability.

[0349] Figures 8 to 15 are focusing on overexpression of activated RAS proteins. RAS proteins are small GTPases, with the most prominent members being HRAS, KRAS and NRAS in humans, which are highly similar to each other. RAS proteins are key regulators in multiple signalling networks, including the PI3K / AKT / mTOR and Ras-Raf-MEK-ERK pathway mentioned above, which means they are also involved in the response of cells to growth factors, hormones and other signals promoting cell growth and proliferation. Due to their prominent role in said pathways, RAS proteins are tightly regulated between an “on” and an ’’off’ state. However, there are known mutations of RAS proteins, especially in the amino acid residues 12,13 and 61 which lock RAS proteins in an constant “on” state and thereby trigger the downstream signalling pathways constantly and independent of upstream signals. Here, we are providing evidence that we can harbour those mutations in cells from agricultural relevant species to remove growth factor and serum requirements of the cells.

[0350] Example 8: Porcine cells overexpressing an activated RAS protein have a growth advantage in growth factor free media

[0351] Methods: Lentiviral vectors were designed to overexpress different activated isoforms of porcine RAS (Seq ID No. 26-31) with a different fluorescent tag (mCERULEAN-KRAS G12D, eGFP-HRAS G12D or mCHERRY-NRAS G12D; Seq ID No. 33-35) under the control of the human EF1A promoter (Seq ID No. 36). The fluorescent tag was separated from the RAS gene by a self-cleaving P2A site (Seq ID No. 32) to allow cleavage of the proteins in translation. Porcine CRISPR immortalized (PSS YRBT7- / HRASG12V / ) myoblasts (see Patent Application PCT / GB2023 / 052528, which is hereby incorporated by reference) with additional edit to aid suspension adaptation (NF2Z) (see Patent Application P44661 GB1). were both infected via 3rdgeneration lentivirus with the RAS overexpression constructs. The multiplicity of infection rate used was 1. For serial passaging, cells were seeded into Erlenmeyer flasks at seeding densities of 5e4-1 e5 cells / mL in 25 mL suspension media with and without 2.5% FBS and with and without a growth factor mix containing 5.25 ng / mL FGF, 8.75 ng / mL IGF1 , 1 ng / mL PDGF0 and 0.035 ng / mL HGF as indicated. Cells were kept in an incubator at 37°C on a shaking platform at 150RPM. Cells were counted and passaged every 3-4 days. For flow cytometry, 5e5-1 e6 cells were sampled at passage 1 / 3 / 4, fixed using 4% PFA and analysed using the MACSQuant® Analyzer 10. Fluorophore expression was measured in live, single cell populations. The channels used to analyse the fluorophores were FITC, VioBlue and PerCP-Vio700 for eGFP (SEQ ID NO: 33), mCERULEAN (SEQ ID NO: 35) and mCHERRY (SEQ ID NO: 34) respectively.

[0352] Results: (a) Schematic design of RAS overexpression constructs used in this study, (b) Experimental design for RAS competition experiment: To determine which RAS isoform promotes cell proliferation the most, porcine myoblast cells overexpressing a RAS G12D isoform coupled to a fluorescent marker (eGFP-HRAS G12D, mCHERRY-NRAS G12D, mCERULEAN-KRAS G12D, indicated in figure by triangle, oval and rectangle respectively) were pooled and grown in triplicates in suspension media with or without growth factors and / or FBS for at least 5 passages. Percentage of cells expressing a given fluorophore in the pooled population were analysed each passage via flow cytometry. In each media condition, a control flask was taken along containing untransduced cells without an activated RAS overexpression to benchmark growth profile, (c) Doubling times (i), cell densities (ii), and % of cells expressing a given fluorophore in cell pool (iii) grown over 6 passages in suspension media containing 2.5% FBS and growth factor mix. (d) Doubling times (i), cell densities (ii), and % of cells expressing a given fluorophore in cell pool (iii) grown over 6 passages in suspension media containing 2.5% FBS but no growth factor mix. (e) Doubling times (i), cell densities (ii), and % of cells expressing a given fluorophore in cell pool (iii) grown over 6 passages in suspension media containing neither growth factor mix nor FBS. Note that control flasks in (d)(i) and (e)(i) did not show cell proliferation in the given media condition, therefore no doubling times were plotted. Note that control flasks were ended after two passages in those conditions as there were not sufficient cells for reseeding as indicated in (d)(ii) and (e)(ii).

[0353] Discussion: Figure 8a shows the general structure of our overexpression constructs that we infect our cells with. We overexpress an activated mutant (G12D) of porcine KRAS, NRAS or HRAS using the strong, constitutively active human EF1A promoter. The proteins are coupled to a fluorescent protein via a P2A self cleaving peptide, which allows tracking or protein expression in a cell pool using flow cytometry without disturbing the function of the RAS proteins. Note that in all following examples, we work with cell pools and not with clonal lines. That means that after infection of cells with a given lentiviral vector, we will have a mix of cells including uninfected cells or cells with one or more copies of the transgene integrated at different positions of the genome.

[0354] Figures 8b-d and 14 shows the results of a competition assay in porcine and bovine cell lines that we used to find out which of the RAS variants shows the best growth effect in those cell lines. For that, we mixed KRAS / NRAS / HRAS cells into a flask and grew that mix in different growth media. Using flow cytometry, we could analyse the % of cells in that mix which express a given Fluorophore-RAS and could therefore draw conclusions which of the RAS variants becomes dominant in the mix over several passages, which means it grows better than the others in the given condition. What we could see e.g. in Figure 8 c / d is that HRAS seems to give a better growth advantage in porcine cells than KRAS and NRAS in media without GF and / or serum. What we also saw though is that in media with GF / serum, all variants deplete, which means that cells seems to take over which contain none of those genes after infection (or where the transgene is not expressed for some reason). This hints that parallel triggering of the signalling pathways by GF / serum and overactivated RAS is detrimental to the cells, although we did not investigate this further, as the actual goal for cultivated meat is growth in media without GF / serum which we could prove here. In contrast, Figure 14 shows that NRAS seems to be the dominant variant in the Wagyu cell line tested. This shows that different RAS isoforms can have different advantages across cell lines. Example 9: Porcine cells overexpressing an activated HRAS protein have a growth advantage in growth factor free media.

[0355] Methods: Porcine myoblast cell pools (P53 / 7RB1 / 7HRASG12V / 7NF2 / ) overexpressing porcine HRAS G12D were grown in suspension media with and without growth factor mix (5.25 ng / mL FGF, 8.75 ng / mL IGF1 , 1 ng / mL PDGF0, 0.035 ng / mL HGF) and with and without 2.5% FBS for 7 passages. Samples were taken at each passage to analyse the percentage of cells expressing eGFP as a proxy for HRAS expressing cells. Growth was compared to untransduced control cells.

[0356] Results: (a) Doubling times (i), cell densities (ii), and % of cells expressing eGFP in cell pool (iii) grown for 7 passages in suspension media containing 2.5% FBS and growth factor mix. (b) Doubling times (i), cell densities (ii), and % of cells expressing eGFP in cell pool (iii) grown for 7 passages in suspension media containing 2.5% FBS but no additional growth factors, (c) Doubling times (i), cell densities (ii), and % of cells expressing eGFP in cell pool (iii) grown for 7 passages in suspension media containing no FBS or growth factors. Note that negative doubling times are not plotted, as e.g. for control line in

[0357] (b)(i) and (c)(i), Also note that control cells died in media without growth factors as seen in (b)(ii) and

[0358] (c)(ii). Note that the % of GFP positive cell population is increasing strongly in growth factor free conditions over the course of the experiment.

[0359] Discussion: Figures 9 and 10 show growth profiles of porcine cells containing just the HRAS or KRAS construct, respectively. Both show growth in all tested media conditions, while control cells only survive in media with GF / serum. Note that similar to the pooled experiment in Figure 8, the fluorophore can only be detected / increase in GF or GF / serum free conditions, while it depletes in full media. Also note that in Figure 9, the initial amount of GFP positive cells was low, which explains the bad growth of the cell pool in the first two passages in the conditions without GF / serum+GF. Only once the HRAS containing cells take over the pool (as shown by the flow cytometry data), the doubling times decrease. Figure 10 also shows that the KRAS cell line has a cell size increase compared to the control cells.

[0360] Example 10: Porcine cells overexpressing an activated KRAS protein have a growth advantage in growth factor free media.

[0361] Methods: Porcine myoblast cell pools (P53 / 7RB1 / 7HRASG12V / 7NF2 / ) overexpressing porcine KRAS G12D were grown in suspension media with 2.5% FBS and with or without growth factor mix (5.25 ng / mL FGF, 8.75 ng / mL IGF1 , 1 ng / mL PDGF0, 0.035 ng / mL HGF) for 6 passages. Samples were taken at each passage to analyse the percentage of cells expressing eGFP as a proxy for KRAS expressing cells and for cell counts. Growth was compared to untransduced control cells.

[0362] Results: (a) Doubling times (i), cell densities (ii) and % of cells expressing mCerulean (iii) grown for 7 passages in suspension media containing 2.5% FBS and growth factor mix. (b) Doubling times (i), cell densities (ii) and % of cells expressing mCerulean (iii) grown for 7 passages in suspension media containing 2.5% FBS of cells and no additional growth factors. Note that negative doubling times are not plotted, as e.g. for control line in (b)(i), also note that control cells died in media without growth factors as seen in (b)(ii). (c) Average cell diameter of untransduced control cells compared to KRAS G12D cells (13.8 pm) compared to control cells (13.1 pm) across all media conditions and passages.

[0363] Example 11 : Porcine cells overexpressing an activated HRAS protein can grow in bioreactors in growth factor and serum free media

[0364] Methods: Porcine myoblast cell pools (P53 / 7RBT / 7HRASG12V / 7NF2 / ) overexpressing porcine HRAS G12D and untransduced control cells were grown in overgrowth studies. For overgrowth studies in flasks, were seeded into 125 mL Erlenmeyer flasks at seeding densities of 2e5 cells / mL in 25mL suspension media with and without a growth factor mix (5.25 ng / mL FGF, 8.75 ng / mL IGF1 , 1 ng / mL PDGFp, 0.035 ng / mL HGF) and with and without 2.5% FBS as indicated. Cells were kept in an incubator at 37°C on a shaking platform at 150 RPM. 500uL samples were taken every day from day 3 onwards to count cells and for spent media analysis. For overgrowth study in the DASbox® Mini Bioreactor System, cells were seeded into reactors at seeding densities of 2.5e5 / mL-3e5 / mL in 160mLs suspension media with and without a growth factor mix (5.25 ng / mL FGF, 8.75 ng / mL IGF1 , 1 ng / mL PDGFp, 0.035 ng / mL HGF) and with and without 2.5% FBS. The cells were kept at 37°C with an agitation rate of 100 RPM. 4mL samples were taken every day to count cells and for spent media analysis. Spent media analysis was performed on Nova Biomedical BioProfile® FLEX2.

[0365] Results: (a) Overgrowth data from cells grown in Erlenmeyer flasks: Cell densities (i), glutamine and ammonia concentrations in spent media (ii), and glucose and lactate concentrations in spent media (iii). (b) Overgrowth data from cells grown in DASbox® Mini Bioreactor System. Cell densities (i), glucose and lactate concentrations in media (ii), glucose and lactate cell usage rates (iii), glutamine and ammonia concentrations in media (iv), glutamine and ammonia cell usage rates (v).

[0366] Discussion: Figures 11 / 13 / 15 shows overgrowth studies (either in flasks or in a DASbox bioreactor system) of HRAS overexpression cells from different animals in various media variants (without GF or without GF and without serum) compared to control cell lines in media with GF / serum. HRAS cells grow well in all conditions, and can sometimes even reach the peak densities of non HRAS cells in full media (Figure 13). Also, nutrient data analysis is given. There seems to be a trend for higher lactate production in cells overexpressing HRAS G12D, while key metabolites Glutamine and ammonia stay mostly unchanged compared to controls.

[0367] Example 12: The growth advantage of cells overexpressing RAS G12D in suspension media without growth factors seen in porcine is consistent in bovine var. Angus cells.

[0368] Methods: Bovine var. Angus CRISPR immortalized (P53 / 7RBT / 7NF2 / ) ADSC were infected with lentivirus containing overexpression constructs for different activated isoforms of porcine RAS with a different fluorescent tag (mCERULEAN-KRAS G12D, eGFP-HRAS G12D or mCHERRY-NRAS G12D). The multiplicity of infection rate used was 1. For serial passaging, cells were seeded separately according to each RAS G12D overexpression construct into Erlenmeyer flasks at seeding densities of 5e4-1 e5 cells / mL in 25 mL suspension media with and without 2.5% FBS and with and without a growth factor mix containing 5.25 ng / mL FGF, 8.75 ng / mL IGF1 , 1 ng / mL PDGFp and 0.035 ng / mL HGF as indicated. Cells were kept in an incubator at 37°C on a shaking platform at 150RPM. Cells were counted and passaged every 3-4 days. Growth was compared to untransduced control cells. For flow cytometry, 5e5-1 e6 cells were sampled at every passage, fixed using 4% PFA and analysed using the MAC SQu a nt® Analyzer 10. Fluorophore expression was measured in live, single cell populations. The channels used to analyse the fluorophores were FITC, VioBlue and PerCP-Vio700 for eGFP, mCERULEAN and mCHERRY respectively.

[0369] Results: (a) Doubling times (i), cell densities (ii) and % of cells expressing each fluorophore (iii) grown in suspension media containing 2.5% FBS and additional growth factor mix. (b) Doubling times (i), cell densities (ii), % of cells expressing each fluorophore (iii) and average doubling times across all 5 passages (iv) grown in suspension media containing 2.5% FBS but no additional growth factors. Average doubling time for Ctrl: 88.1 h, for HRAS G12D: 26.4 h, for KRAS G12D: 31.2 h, for NRAS G12D: 32.1 h. (c) Doubling times (i), cell densities (ii), % of cells expressing each fluorophore (iii) and average doubling times across all 5 passages (iv) grown in suspension media without FBS or additional growth factors. Average doubling time for HRAS G12D: 29.3 h, for KRAS G12D: 37.2 h, for NRAS G12D: 39.4 h. Note that negative doubling times are not plotted, as e.g. for control line in (c)(i), also note that control cells died in media without growth factors as seen in (c)(ii).

[0370] Discussion: Figure 12 shows growth profiles of bovine var. Angus ADSC containing HRAS, KRAS or NRAS overexpression constructs and control cells respectively. Similarly to the porcine cells, only RAS overexpression cells can grow in media without GF or without GF / serum and HRAS overexpression seems to have the strongest effect as seen in doubling times comparison and also from flow cytometry enrichment.

[0371] Example 13: Bovine var. Angus cells overexpressing an activated HRAS protein show comparable growth in media without growth factors as control cells in media with growth factors in overgrowth studies

[0372] Methods: Bovine var. Angus CRISPR immortalized (P53 / 7RBT / 7NF2 / ) ADSC pools overexpressing HRAS G12D were seeded into 125 mL Erlenmeyer flasks at seeding densities of 1 e5 cells / mL in 25mL suspension media with and without a growth factor mix (5.25 ng / mL FGF, 8.75 ng / mL IGF1 , 1 ng / mL PDGFp, 0.035 ng / mL HGF) and with and without 2.5% FBS as indicated. Growth was compared to untransduced control cells. Cells were kept in an incubator at 37°C on a shaking platform at 150 RPM for seven days. 500uL samples were taken every day from day 3 onwards to count cells and for spent media analysis. Spent media analysis was performed on Nova Biomedical BioProfile® FLEX2.

[0373] Results: (a) Cell densities (i), glutamine and ammonia levels in the media (ii), glucose and lactate levels in the media (iii), glutamine and ammonia cell usage rates (iv) and glucose and lactate cell usage rates (v) over 7 days growth period.

[0374] Discussion: Figure 14 shows growth data from a mix of RAS G12D Wagyu cells (described above) but also from a Wagyu cell pool overexpressing HRAS G12D only. Similar conclusion as beforehand, only RAS overexpressing cells survive in media without GF / serum+GF. One difference to other cell lines is that RAS seems to give a slight growth advantage also in full media conditions, as can be seen on the doubling times as well as on the stable expression of the transgene in this condition (porcine / Angus cells showed depletion of the fluorescent cells in full media conditions). Again, we see a cell size increase in RAS overexpressing cells (14e).

[0375] Example 14: Bovine var. Wagyu cells overexpressing an activated RAS protein have a growth advantage in growth factor free media

[0376] Methods: Bovine var. Wagyu CRISPR immortalized (P53 / 7RBT / 7NF2 / ) ADSC were infected with lentivirus containing overexpression constructs for different activated isoforms of porcine RAS with a different fluorescent tag (mCERULEAN-KRAS G12D, eGFP-HRAS G12D or mCHERRY-NRAS G12D). The multiplicity of infection rate used was 1 . For serial passaging, cells expressing different RAS G12D isoforms were pooled into Erlenmeyer flasks at seeding densities of 5e4-1 e5 cells / mL and grown in triplicates in 25 mL suspension media with and without 2.5% FBS and with and without a growth factor mix containing 5 ng / mL FGF, 10 ng / mL IGF1 , 1 ng / mL PDGF0 and 0.1 ng / mL HGF as indicated. Similarly, three flasks were grown in those conditions containing only cells transduced with the HRAS G12D overexpression construct. Growth was benchmarked against a flask with untransduced control cells. Cells were kept in an incubator at 37°C on a shaking platform at 150RPM. Cells were counted and passaged every 3-4 days. Doubling time and cell size were compared. For flow cytometry, 5e5-1 e6 cells were sampled at every passage, fixed using 4% PFA and analysed using the MACSQuant® Analyzer 10. Fluorophore expression was measured in live, single cell populations. The channels used to analyse the fluorophores were FITC, VioBlue and PerCP-Vio700 for eGFP, mCERULEAN and mCHERRY respectively.

[0377] Results: (a) Doubling times (i), cell densities (ii), % of cells expressing a given fluorophore in the pool (iii) and % of cells expressing eGFP in cells overexpressing HRAS G12D grown separately (iv) grown for 5 passages in suspension media containing 2.5% FBS and growth factor mix. (b) Doubling times (i), cell densities (ii), % of cells expressing a given fluorophore in the pool (iii) and % of cells expressing eGFP in cells overexpressing HRAS G12D grown separately (iv) grown for 5 passages in suspension media containing 2.5% FBS but no additional growth factors, (c) Doubling times (i), cell densities (ii), % of cells expressing a given fluorophore in the pool (iii) and % of cells expressing eGFP in cells overexpressing HRAS G12D grown separately (iv) grown for 5 passages in suspension media containing no FBS or growth factors. Note that negative doubling times are not plotted, as e.g. for control line in (c)(i), Also note that control cells died in media without FBS and growth factors as seen in (c)(ii). (d) Average doubling times across the passages of the assay comparing the control cells, the pooled cells and the eGFP-HRAS G12D cells alone in suspension media containing 2.5% FBS and additional growth factor mix (i), with Ctrl: 33.5 h, HRAS G12D: 30.9 h, Pool: 29.2 h; in suspension media with 2.5% FBS but no additional growth factors (ii), with Ctrl: 152.3 h, HRAS G12D: 31 h, Pool: 29.9 h; and in suspension media without FBS or additional growth factors (iii), with HRAS G12D: 30.1 h, Pool: 32.6 h;. Note that data from passage 3 is not included in the average doubling times, (e) Average cell diameter of untransduced control cells compared to HRAS G12D cells compared across all media conditions and passages, with Ctrl: 14.5 pm, HRAS G12D: 16.2 pm.

[0378] Example 15: Bovine var. Wagyu cells overexpressing an activated HRAS protein show growth in media without growth factors as control cells in media with growth factors in overgrowth studies Methods: Bovine var. Wagyu CRISPR immortalized (P53 / 7RBT / 7NF2 / ) ADSC pools overexpressing HRAS G12D were seeded into 125 mL Erlenmeyer flasks at seeding densities of 1 e5 cells / mL in 25mL suspension media with and without a growth factor mix (5 ng / mL FGF, 10 ng / mL IGF1 , 1 ng / mL PDGF0, 0.1 ng / mL HGF) and with and without 2.5% FBS as indicated. Growth was compared to untransduced control cells for seven days. Cells were kept in an incubator at 37°C on a shaking platform at 150 RPM. 500uL samples were taken every day from day 3 onwards to count cells and for spent media analysis. Spent media analysis was performed on Nova Biomedical BioProfile® FLEX2.

[0379] Results: (a) Cell densities (i), glutamine and ammonia levels in spent media (ii), glucose and lactate levels in the media (iii), glutamine and ammonia cell usage rates (iv) and glucose and lactate cell usage rates (v) over 7 days growth period.

[0380] Discussion: Figures 16 and 17 try to tie the PTEN / SPRY2 and RAS stories together by knocking out PTEN / SPRY2 in the HRAS G12D overexpression Wagyu cell line. We can show that the editing is successful and retained over time. We also show that there is not much change in growth of those cells compared to HRAS only cell lines (in some conditions slight disadvantage but seems more or less the same). However, we show that we can boost the cell size even further when combining both strategies. In Figure 17, we additionally show that we can remove insulin (which was part of the suspension media in all previous experiments) from RAS overexpression cells and RAS overexpression cells with a PTEN / SPRY2 KO in an overgrowth study without much negative effect. This shows that removal of insulin, another expensive media component is possible using those modifications.

[0381] Figures 16 and 17 are focusing on combining the overexpression of activated RAS proteins with a knockout in the PTEN / SPRY2 genes.

[0382] Example 16: The PTEN / SPRY2 double knockout can be combined with an HRAS G12D overexpression in bovine var. Angus ADSC and leading to an additional cell size increase.

[0383] Methods: Bovine var. Wagyu CRISPR immortalized (P53 / 7RB1 / 7NF2 / ) ADSC overexpressing a porcine HRAS G12D protein (eGFP-HRAS G12D) were edited using nucleofection with synthetic single guide RNAs (Seq ID No. 20, Seq ID No. 22) against the bovine PTEN and SPRY2 genes (Seq ID No. 5, Seq ID No. 13) and Strep. Pyogenes Cas9 protein. Editing efficiencies were measured at two time points post editing to screen for enrichment or depletion of desired mutations. Enrichment of a mutation of interest suggest a positive impact of a given mutation on cell growth, while depletion suggests a detrimental effect on cell health or growth. Editing efficiencies were measured by PCR amplification of target region, Sanger Sequencing and Synthego ICE analysis of the Sanger Sequencing file (https: / / ice.sy thego.eom / # / ). Note that cells were co-edited with sgRNAs against the P53 and RB1 genes to immortalize the cell lines (see Patent Application PCT / GB2023 / 052528, which is hereby incorporated by reference) and the NF2 gene (see Patent Application P44661 GB1) to reduce cell doubling time in suspension.

[0384] For serial passaging, cells were seeded into 125 mL Erlenmeyer flasks at seeding densities of 5e4-1 e5 cells / mL and grown in triplicates in 25 mL suspension media with and without 2.5% FBS as indicated. Growth of HRAS G12D / PTEN( / ) / SPRY2( / )lines was benchmarked against HRAS G12D / PTEN<+ / +) / SPRY2<+ / +) lines. Cells were kept in an incubator at 37°C on a shaking platform at 150RPM. Cells were counted and passaged every 3-4 days. Doubling time and cell size were compared. Results: (a) (b) (i) doubling times (ii) cell densities, (iii) pooled doubling times over all 4 passages and (iv) pooled cell size of bovine var. Wagyu adipose derived stem cell (ADSC) pools in suspension media containing 2.5% FBS and no growth factor. Average doubling time for the Ctrl is 41 .5 h and 42 h for the PTEN <- / ) / SPRY2 w cells. Average cell size for the Ctrl (PTEN <+ / +> / SPRY2 <+ / +>) is 16.1 pm and 16.5 pm for PTEN( / ) / SPRY2( / )cells, (c) (i) doubling times (ii) cell densities, (iii) pooled doubling times over all 4 passages and (iv) pooled cell size of bovine var. Wagyu adipose derived stem cell (ADSC) pools in suspension media containing no FBS and no growth factor. Average doubling time for the Ctrl is 37.2 h and 44.3 h for the PTEN < / ) / SPRY2 cells. Average cell size for the Ctrl (PTEN <+ / +> / SPRY2 <+ / +>) is 16.1 pm and 16.7 pm for PTEN<- / -) / SPRY2<- / -) cells.

[0385] Discussion: We can show that the editing both genes in a cell line overexpressing HRAS G12D is successful and retained over time. We also show that while growth of those cells compared to HRAS only cell lines is comparable, we can boost the cell size even further when combining both strategies.

[0386] Example 17: The PTEN / SPRY2 double knockout can be combined with an HRAS G12D shows similar growth to HRAS G12D overexpression lines in an overgrowth study and both cell lines grow in media containing no FBS, no growth factors and no insulin.

[0387] Methods: Bovine var. Angus CRISPR immortalized (P53 / 7RBT / 7NF2 / ) ADSC pools overexpressing HRAS G12D and containing a PTEN / SPRY2 double knockout (PTEN( / ) / SPRY2( / )) were seeded into 125 mL Erlenmeyer flasks at seeding densities of 1 e5 cells / mL in 25mL suspension media without FBS and without growth factor mix as well as without 2.5% FBS, without growth factor mix and without insulin as indicated. Growth was compared to control cells without the PTEN / SPRY2 double knockout but otherwise identical genotype (RAS G12D / P53- / 7RBT / 7NF2 / 7PTEN(+ / +) / SPRY2<+ / +)) for seven days. Cells were kept in an incubator at 37°C on a shaking platform at 150 RPM. 500uL samples were taken every day from day 3 onwards to count cells

[0388] Results: (a) (i) Viable cell densities, (ii) cell diameter) and (iii) cell viability of cells growing in suspension media without FBS and GF but containing insulin, (b) (i) Viable cell densities, (ii) cell diameter) and (iii) cell viability of cells growing in suspension media without FBS, GF, and insulin. Discussion: In this example, we additionally show that we can remove insulin in the suspension media from RAS overexpression cells and RAS overexpression cells with a PTEN / SPRY2 KO in an overgrowth study without much negative effect. This shows that removal of another expensive media component is possible using those modifications.

[0389] Example 18: BAX and BAK1 can be edited efficiently in Wagyu muscle-derived cells and edits are retained over time.

[0390] Methods: Wagyu muscle-derived cells were CRISPR edited using nucleofection with synthetic single guide RNAs and Strep. Pyogenes Cas9 protein. Target genes were P53, RB1 , NF2 and PTEN for the control cell line, and P53, RB1 , NF2, PTEN, BAX and BAK for the BAX- / -BAK1 - / - cell line. Editing efficiency was assessed by PCR amplification of the target region followed by Sanger Sequencing, at day 3 and day 10 post-editing. Sequencing data was analysed using Synthego ICE. Edits were considered a knockout (KO) if they contain a frameshift mutation or indels of 21 + base pairs, Other Indels refers to percentage of sequences that contain an indel which does not lead to a frameshift, WT refers to percentage of sequences that are wild type, unknown refers to the percentage of Sanger sequencing reads that cannot be explained by the ICE analysis tool.

[0391] Results: Editing data for Figure 18 (a) the control cell line and (b) the BAX- / -BAK1- / - cell line demonstrate high knockout scores for P53, RB1 , NF2 and PTEN at both Day 3 and Day 10. BAX and BAK were also knocked out with high efficiency in the BAX- / -BAK1- / - cell line. All edits were retained over time, between day 3 and day 10. Sequencing data for Day 3 BAK1 samples was poor quality and therefore not included in the analysis.

[0392] Example 19: Wagyu muscle-derived cells containing a BAX / BAK1 double knockout maintain peak cell density for longer and have a larger cell diameter in a suspension culture overgrowth study.

[0393] Methods: Cells were seeded into triplicate 125 mL Erlenmeyer flasks at a density of 1 e6 cells / mL in serum-free suspension media. The growth of BAX- / -BAK1- / - cells was compared to a BAX+ / +BAK1 + / + control cell line (Ctrl). Cells were counted every 24 hours from day 1 to 10 to calculate (a) viable cell density (b) cell diameter. Average cell diameter over the course of the experiment was also determined (c) and analysed for statistical significance using a Wilcoxon matched-pairs signed rank test.

[0394] Results: Figure 19 (a) Cells containing a BAX / BAK1 double knockout maintained a peak viable cell density of around 4.6e6 cells / mL from day 6 to 9, whilst the cell density of the control cell line decreased during this time, (b) Cells with a BAX / BAK1 double knockout had a larger diameter from day 4 to 10. (c) Average diameter over 10 days was significantly larger in BAX- / -BAK1- / - cells (14 pM) compared to the control cell line (13.4 pM).

[0395] Example 20: Wagyu muscle-derived cells containing a BAX / BAK1 double knockout maintain peak cell density for longer and have a larger cell diameter in a suspension culture overgrowth study. Methods: Cells were seeded into triplicate 125 mL Erlenmeyer flasks at a density of 1 e6 cells / mL in serum-free suspension media. The growth of BAX- / -BAK1- / - cells was compared to a BAX+ / +BAK1 + / + control cell line (Ctrl). Cells were counted every 24 hours from day 1 to 8 to calculate (a) viable cell density (b) cell diameter. Average cell diameter over the course of the experiment was also determined (c) and analysed for statistical significance using a Wilcoxon matched-pairs signed rank test. On day 7, cells were stained with annexin v and fluorescence was measured on the MACSQuant 10 flow cytometer to assess the number of apoptotic cells. A negative control sample was included, which were cells in exponential phase of growth, which would not be expected to be apoptotic.

[0396] Results: Figure 20 (a) Cells containing a BAX / BAK1 double knockout maintained a peak viable cell density of around 5.3e6 cells / mL from day 5 to 7, whilst the cell density of the control cell line decreased during this time, (b) Cells with a BAX / BAK1 double knockout had a larger diameter from day 4 to 7. (c) Average diameter over 10 days was significantly larger in BAX- / -BAK1- / - cells (13.6 pM) compared to the control cell line (13.1 pM). (d) ); Annexin v staining demonstrated that there were fewer apoptotic cells present at Day 7 in BAX- / -BAK1- / - cells (22%) compared to the control cell line (53%), when gated relative to the negative control sample.

[0397] Example 21 : Wagyu muscle-derived cells containing a BAX / BAK1 double knockout have improved apoptosis resistance in a suspension culture overgrowth study

[0398] Methods: Cells were seeded into triplicate 125 mL Erlenmeyer flasks at a density of 1 e6 cells / mL in serum-free suspension media. The growth of BAX- / -BAK1- / - cells was compared to a BAX+ / +BAK1 + / + control cell line (Ctrl). Cells were counted every 24 hours from day 4 to 7 to calculate (a) viable cell density and (b) cell diameter. On day 7, cells were stained with propidium iodide (PI) and annexin v and fluorescence was measured on the MACSQuant 10 flow cytometer to assess the number of dead and apoptotic cells respectively.

[0399] Results: Figure 21 (a) Cells containing a BAX / BAK1 double knockout maintained a viable cell density of around 4.9e6 cells / mL from day 5 to 7, whilst the cell density of the control cell line decreased during this time, (b) Cells with a BAX / BAK1 double knockout had a larger diameter, (d) Whilst viability was similar between the two cell lines (30% PI positive cells in the control cell line vs 23% in BAX- / -BAK1- / - cells); annexin v staining demonstrated that there were fewer apoptotic cells present at Day 7 in BAX- / - BAK1- / - cells (16%) compared to the control cell line (45%).

[0400] Discussion of results from Examples 18-21 :

[0401] Apoptotic cell death poses a significant challenge in large-scale cell culture systems, such as bioreactors used for cultivated meat production, as it leads to reduced productivity and lower overall yield. Apoptosis can be triggered by nutrient depletion at the end of batch culture, as well as stress factors like increased osmolality and shear stress.

[0402] In this study, we demonstrate that double knockout of BAX and BAK1 in Wagyu muscle-derived cells leads to sustained peak cell density over an extended period and a significantly larger cell diameter. The increase in cell size is attributed to reduced apoptotic activity, as apoptotic cells typically undergo shrinkage, which we confirmed using annexin V staining. These advantages are particularly relevant for cultivated meat production, as larger cells contribute more biomass per cell, improving overall yield and product quality. When scaling up to industrial bioreactors, even small increases in cell size can have a substantial impact on total biomass output.

[0403] Sequences

Claims

Claims1 . A method of culturing an animal cell comprising culturing the animal cell in a cell culture medium, wherein the cell culture medium is serum, exogenous growth factor, and / or insulin free or wherein the cell culture medium is reduced in serum, at least one exogenous growth factors, and / or insulin, and wherein the animal cell comprises a genetic modification in one or more of a RAS gene, the TP53 gene, the RB1 gene, the NF2 gene, the PTEN gene, the SPRY gene, the BAX gene and / or the BAK1 gene.

2. The method of claim 1 , wherein the cell culture medium comprises 2.5% serum or less.

3. The method according to any one of claims 1 or claims 2, wherein the cell culture medium comprises at least one exogenous growth factors at a concentration of 20 ng / mL or less.

4. The method according to any one of claims 2 to 3, wherein the cell culture medium comprises 100 pg / mL insulin or less.

5. The method of any one of the preceding claims, wherein the animal is of an animal species suitable for human or animal consumption.

6. The method of claim 5, wherein the animal is selected from a pig, bovine, poultry, sheep, goat, fish, Camelidae, Equidae, crustaceans or mollusc.

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

8. The method according to any one of the preceding claims, wherein the animal cell is selected from one of the following cell types: myoblast, fibroblast, myofibroblast, adipose derived stem cell, epithelial cell, mesenchymal stem cell, satellite cell, iPSC, or hepatocyte.

9. The method according to any one of the preceding claims, wherein the genetic modification in the animal cell is by any one or more of:1) gene level modification by: a. knock-out or reduced activity / transcription / translation levels via editing in coding sequences, promoters, introns, regulatory regions; b. RNA-directed DNA methylation; or c. transcription activation or repression using CRISPRa or CRISPRi or similar target specific methods;d. knock-out or reduced activity / transcription / translation levels via undirected means, for example radiation or chemical mutagenesis; e. overexpression of an endogenous nucleotide sequence2) post-transcription level (post-transcriptional gene silencing) modification by: a. RNAi or siRNA to reduce translation of mRNA into protein; or b. site specific nucleases to modify or cleave mRNA, such as CRISPR / Cas13a;3) post-translational level (protein disruption or activation) modification by: a. inclusion of activity blocking / reducing or enhancing molecules, wherein the activity blocking / reducing or enhancing molecules are small molecules, antibodies, or the like; or b. inclusion of protein degrading ingredients, wherein the protein degrading ingredients are specialised proteases, exoproteases, or endoproteases. c. Enhancing or reducing of protein activity through integration of activating or reducing functional mutations in the corresponding gene sequences.

10. The method according to any one of the preceding claims, wherein the modification decreases 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%, or 95% or about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7- fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold or 100-fold in comparison to the unmodified animal cell.

11. The method according to any one of the preceding claims, wherein the modification increases cell size by at least about 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150% or 200% or about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold or 100- fold in comparison to the unmodified animal cell.

12. The method according to any one of the preceding claims, wherein the genetic modification is a loss of function modification or leads to reduction in function, wherein the loss of function modification comprises a knock-out of the gene or loss of protein function.

13. The method according to any one of the preceding claims, wherein the modification is introduced using targeted genome modification or randomised mutagenesis or by spontaneous mutation.

14. The method according to any one of the preceding claims, wherein the modification is in the promoter region or coding region of one or more genes.

15. The method according to any one of the preceding claims, wherein the modification is introduced using targeted genome modification, optionally using a targeted endonuclease.

16. The method according to claim 15, wherein the endonuclease is selected from TALEN, ZFN or CRISPR, optionally CRISPR / Cas9.

17. The method according to any one of the preceding claims, wherein the animal cell comprises a genetic modification in a RAS gene, the PTEN gene and / or a SPRY gene.

18. The method according to any one of the preceding claims, wherein the SPRY gene is selected from SPRY1, SPRY2, SPRY3, and / or SPRY4.

19. The method according to claim 18, wherein the SPRY gene is SPRY2.

20. The method according to any one of the preceding claims, wherein the genetic modification in the TP53 gene, the RB1 gene, the NF2 gene, the PTEN gene and / or a SPRY gene is a loss of function modification.21 . The method according to claim 20, wherein the loss of function modification comprises a knockout of the gene.

22. The method according to claim 20 or claim 21 , wherein the genetic modification in the TP53 gene, the RB1 gene, the NF2 gene, the PTEN gene and / or a SPRY gene is a knock out genetic modification in one or both alleles.

23. The method according to any one of the preceding claims, wherein the animal cell also has a genetic modification in a RAS gene.

24. The method according to claim 23, wherein the RAS gene is HRAS, NRAS, or KRAS.

25. The method according to claim 24, wherein the RAS gene is HRAS.

26. The method according to any one of claims 23 to 25, wherein the genetic modification to theRAS gene is an overexpression and / or hyperactivation of the gene or gene product.

27. The method according to any one of the preceding claims, wherein the method comprises continuous or batch culture of the modified animal cell.

28. The method according to any one of the preceding claims, wherein the method comprises the step of forming the animal cells into a tissue like structure.

29. The method according to claim 28, wherein the animal cells are formed into a muscle tissue like structure.

30. The method according to any one of claims 27 to 29, wherein the modified animal cell is used in the production of cultivated meat or a cultured meat product.

31. A cultured animal cell for use in the method of any one of claims 1 to 30.

32. A cultured animal cell according to claim 31 , wherein the cultured animal cell comprises a genetic modification in the PTEN gene and / or SPRY gene and wherein the animal is of an animal species suitable for human or animal consumption.

33. The cultured animal cell according to claim 32, wherein the SPRY gene is selected from a group consisting of SPRY1, SPRY2, SPRY3, and / or SPRY4.

34. The cultured animal cell according to claim 32 or claim 33, wherein the SPRY gene is SPRY2.

35. The cultured animal cell according to any one of claims 31 to 34, wherein the animal is selected from a pig, bovine, poultry, sheep, goat, Equidae, Camelidae, fish, crustaceans or mollusc.

36. The cultured animal cell according to any one of claims 31 to 35, wherein the animal cell is a somatic cell.

37. The cultured animal cell according to any one of claims 31 to 36, wherein the animal cell is selected from one of the following cell types: myoblast, fibroblast, myofibroblast, adipose derived stem cell, epithelial cell, mesenchymal stem cell, satellite cell, iPSC, or hepatocyte.

38. The cultured animal cell according to any one of claims 31 to 37, wherein the genetic modification in the PTEN gene and / or SPRY gene is a loss of function modification or leads to reduction in function.

39. The cultured animal cell according to claim 38, wherein the loss of function modification comprises a knock-out of the PTEN gene and / or SPRY gene or loss of protein function.

40. The cultured animal cell according to any one of claims 31 to 39, wherein the modification is introduced using targeted genome modification or randomised mutagenesis or by spontaneous mutation.

41. The cultured animal cell according to any one of claims 31 to 40, wherein the modification is in the promoter region or coding region of one or more genes.

42. The cultured animal cell according to any one of claims 31 to 41 , wherein the modification is introduced using targeted genome modification, optionally using a targeted endonuclease.

43. The cultured animal cell according to claim 42, wherein the endonuclease is selected from meganuclease, TALEN, ZFN or CRISPR, optionally CRISPR / Cas9.

44. A method of producing cultivated meat or a cultured meat product comprising culturing the animal cell according to any one of claims 31 to 43.

45. The method of producing the cultured animal cell according to any one of claim 44, wherein the modification decreases the doubling time and / or increases cell size 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%, or 95% in comparison to the unmodified animal cell or about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8- fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold or 100-fold in comparison to the unmodified animal cell..

46. Cultivated or cultured animal tissue or a cultivated or cultured meat product comprising a modified cell according to any one of claims 31 to 43.

47. Use of the modified cultured animal cell according to any one of claims 31 to 43 for cellular agriculture.

48. A guide RNA targeting the sequence for the PTEN gene or the sequence for the SPRY gene in a cultured animal cell according to any of claims 31 to 43.

49. The guide RNA according to claim 48, wherein the guide RNA targets the sequence for the PTEN gene and comprises a sequence according to SEQ ID NO: 19 or SEQ ID NO: 20.

50. The guide RNA according to claim 48, wherein the guide RNA targets the sequence for the SPRY1 gene and comprises a sequence according to SEQ ID NO: 21 .51 . The guide RNA according to claim 50, wherein the guide RNA targets the sequence for the SPRY2 gene and comprises a sequence according to SEQ ID NO: 22 or SEQ ID NO: 23.

52. The guide RNA according to claim 51 , wherein the guide RNA targets the sequence for theSPRY3 gene and comprises a sequence according to SEQ ID NO: 24.

53. The guide RNA according to claim 52, wherein the guide RNA targets the sequence for theSPRY4 gene and comprises a sequence according to SEQ ID NO: 25.

54. A kit comprising at least one guide RNA of any one of claims 48 to 53.

55. A modified animal cell comprising a genetic modification in the BAK1 gene and / or the BAX gene, and wherein the animal is of an animal species suitable for human or animal consumption.

56. The modified animal cell according to claim 55, wherein the animal is selected from a pig, bovine, poultry, sheep, goat, Equidae, Camelidae, fish, crustaceans or mollusc, preferably wagyu bovine cells.

57. The modified animal cell according to any one of claims 55 to 56, wherein the animal cell is a somatic cell.

58. The modified animal cell according to any one of claims 55 to 57, wherein the animal cell is selected from one of the following cell types: myoblast, fibroblast, myofibroblast, adipose derived stem cell, epithelial cell, mesenchymal stem cell, satellite cell, iPSC, or hepatocyte.

59. The modified animal cell according to any one of claims 55 to 58, wherein the genetic modification is a gene is a loss of function modification or leads to reduction in function.

60. The modified animal cell according to any one of claims 55 to 59, wherein genetic modification comprises a knock-out of the BAK1 gene and / or the BAX gene or loss of protein function.61 . The modified animal cell according to any one of claims 55 to 60, wherein the modification is introduced using targeted genome modification or randomised mutagenesis or by spontaneous mutation.

62. The modified animal cell according to any one of claims 55 to 61 , wherein the modification is in the promoter region or coding region of one or more genes.

63. The modified animal cell according to any one of claims 55 to 62, wherein the modification is introduced using targeted genome modification, optionally using a targeted endonuclease.

64. The modified animal cell according to any one of claims 55 to 63, wherein the endonuclease is selected from meganuclease, TALEN, ZFN or CRISPR, optionally CRISPR / Cas9.

65. A method of producing cultivated meat or a cultured meat product comprising culturing the modified animal cell according to any one of claims 55 to 64.66 A method of conferring apoptosis resistance, improving cell viability, increasing cell density, increasing cell size and / or increasing cell lifespan of a cultivated or cultured animal cell suitable for human or animal consumption comprising cultivating or culturing animal cells comprising a genetic modification in the BAK1 gene and / or the BAX' gene.

67. The method of producing the modified animal cell according to any one of claim 65 or the method of conferring apoptosis resistance, improving cell viability, increasing cell density, increasing cell size and / or increasing cell lifespan of a cultivated or cultured animal cell suitable for human or animal consumption according to claim 66, wherein the modification reduces apoptosis, increases viability, increases density, increases cell size and / or increases lifespan 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%, or 95% in comparison to the unmodified animal cell, or about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9- fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold or 100-fold in comparison to the unmodified animal cell.

68. Use of the modified cultured animal cell according to any one of claims 55 to 64 for cellular agriculture.

69. A guide RNA targeting the sequence for the BAK1 gene or the sequence for the BAX gene in a cultured animal cell according to any of claims 55 to 64.

70. The guide RNA according to claim 69, wherein the guide RNA targets the sequence for the BAK1 gene and comprises a sequence according to SEQ ID NO. 4271 . The guide RNA according to claim 69, wherein the guide RNA targets the sequence for the BAX gene and comprises a sequence according to SEQ ID NO. 41 .

72. A kit comprising at least one guide RNA of any one of claims 69 to 71 .

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