NZ794568A - Controllable transcription - Google Patents

Abstract

The present invention relates to a stable method for introducing at least one inducible cassette into a cell, and permitting controllable transcription from within that inducible cassette. The method may be used for any cell type, from any eukaryotic organism, but has a particular application in the introduction of inducible cassettes into pluripotent stem cells, such as animal or human pluripotent stem cells (hPSCs). The inducible cassette is controllably inserted in such a way to ensure that the genetic material it contains is not silenced or subject to negative influences from the insertion site, and transcription of the genetic material is controlled. introduction of inducible cassettes into pluripotent stem cells, such as animal or human pluripotent stem cells (hPSCs). The inducible cassette is controllably inserted in such a way to ensure that the genetic material it contains is not silenced or subject to negative influences from the insertion site, and transcription of the genetic material is controlled.

Description

The present invention relates to a stable method for introducing at least one inducible cassette into a cell, and permitting controllable transcription from within that inducible cassette. The method may be used for any cell type, from any otic organism, but has a particular application in the introduction of ble cassettes into pluripotent stem cells, such as animal or human pluripotent stem cells (hPSCs). The inducible cassette is controllably inserted in such a way to ensure that the genetic material it contains is not silenced or subject to negative nces from the insertion site, and transcription of the genetic material is controlled.

NZ 794568 Controllable transcription Cross reference to related applications This application is a divisional of New Zealand Application No 753732 which is the New Zealand national phase entry of , which claims priority to GB 1619876.4 filed 24 November 2016. Each of these ations is herein orated by nce in their entireties.

The present invention relates to a stable method for introducing at least one inducible cassette into a cell, and permitting controllable transcription from within that inducible cassette. The method may be used for any cell type, from any eukaryotic organism, but has a particular application in the introduction of inducible tes into pluripotent stem cells, such as animal or human pluripotent stem cells (hPSCs). The inducible cassette is controllably inserted in such a way to ensure that the c material it contains is not silenced or subject to negative influences from the insertion site, and ription of the genetic material is controlled.

Background to the Invention Stem cell ch holds great promise for research of human development, regenerative medicine, disease modelling, drug discovery, and cell transplantation. Moreover, stem cell-derived cells enable studying physiological and pathological responses of human cell populations that are not easily ible. This often entails the study of genes (and other forms of regulatory mechanisms encoded in non-protein-coding RNAs - ncRNAs). Unfortunately, controllable transcription or expression of genetic information in human cells has been proven to be particularly difficult. er, for several key aspects of regenerative medicine, disease modelling, drug discovery and cell transplantation, manipulation and manufacture of mature human cell types from easily accessible sources is required. Controlling the expression of transgenes in human cells is the basis of biological research. However, this has proven to be difficult in human cells. Moreover, there is a real need for the in vitro derivation of many highly desirable human cell types in a quantity and quality suitable for drug discovery and regenerative medicine purposes. Because directed differentiation of stem cells into desired cell types is often challenging, other ches have emerged, including direct reprogramming of cells into the desired cell types. In particular, forward programming, as a method of directly converting otent stem cells, including hPSCs, to mature cell types has been recognised as a powerful strategy for the derivation of human cells. This reprogramming involves the forced expression of key lineage transcription factors (or non-coding RNAs, including lncRNA and microRNA) in order to convert the stem cell into a particular mature cell type. Also in this context, controllable expression of genetic information in human cells has been challenging. Currently available d programming protocols are largely based on lentiviral transduction of cells, which s in variegated expression or te silencing of randomly inserted inducible cassettes. This results in additional purification steps in order to isolate a pulation sing the required ription factors. Thus, further refinements of these methods are y required.

Apart from inducible expression of transgenes, it is very desirable to be able to control knockdown and knockout of genes or other coding sequences in cells, to allow loss of function studies to be carried out. Loss of on studies in stem cells and mature cell types provide a unique opportunity to study the isms that regulate human pment, disease and logy. However, t techniques do not permit the easy and efficient manipulation of gene expression. The current techniques to introduce material such as inducible short hairpin RNAs (shRNA) into stem cells to trigger gene knockdown suffer from many of the drawbacks seen with the forward ramming sed above, such as transgene ing and positional effects limiting activity. Thus, there is a need for inducible gene knockout and knockdown in stem cells that allows for loss of function studies in stem cells.

Any refinements to the above methods must ensure that stable transcription of the genetic material contained within the inducible cassette, such as a transgene, is achieved which is resistant to silencing and other negative integration site-related influences. Silencing may be caused by multiple epigenetic mechanisms, including DNA methylation or histone modifications. With prior art methods based on lentiviral transduction, the cells obtained are a heterogeneous population with the transgene expressed fully, partially or silenced. Clearly, this is not desirable for many applications. Viral vectors demonstrate a tendency to integrate their genetic material into transcriptionally active areas of the genome, thus increasing the potential for oncogenic events due to insertional mutagenesis.

For many ations, it is desirable to control the ription of inserted genetic material in a cell, such that an inducible cassette may be turned on as required and transcribed at particular levels, including high levels.

This cannot be achieved if the insertion of the inducible cassette is random in the genome.

The inventors have thus ped a method for enabling the stable introduction of an inducible cassette into the genome of a cell, whilst being able to control the transcription of that inducible cassette. This has benefits in any cell type in which it is d to introduce an inducible cassette and control transcription of the inserted genetic material, in particular in pluripotent stem cells. The inducible cassette may e any genetic material capable of transcription, for example a transgene or a non-coding RNA (ncRNA). The al included within the inducible cassette will be determined by what effects are required from the stem cell, including expression of a ene or gene knockdown or knockout.

Summary of the Invention The inventors have found that it is possible to insert an inducible cassette and control transcription of the genetic material within that inducible cassette by using a dual genomic safe harbour targeted system herein described. Such a method is highly desirable, since there is d risk of epigenetic silencing of the inserted genetic al, and it is possible to obtain a homogenous population of cells transcribing the ble cassette.

In a first aspect, the invention relates to an ex vivo method for controlling transcription of a genetic sequence in a cell comprising: a) targeted insertion of a gene encoding a transcriptional regulator protein into a first genetic safe harbour site; and b) targeted insertion of an inducible cassette into a second genetic safe r site; wherein said inducible cassette comprises said genetic sequence operably linked to an inducible promoter, and said promoter is regulated by the transcriptional regulator protein; wherein said first and second genetic safe harbour sites are different; and n the cell is an animal cell.

In a second , the invention relates to an ex vivo method for the production of myocytes from pluripotent stem cells, sing the steps of: a) ed insertion of a gene encoding a transcriptional regulator protein into a first genetic safe harbour site; and b) targeted insertion of the MYOD1 gene operably linked to an inducible promoter into a second genetic safe harbour site; and c) culturing said cells in the presence of retinoic acid; wherein said ble promoter is regulated by the transcriptional regulator protein; wherein said first and second genetic safe harbour sites are different; and wherein the pluripotent stem cell is an animal cell.

In a third aspect, the invention s to a cell obtainable by the method according to the first aspect or the second aspect.

In a fourth aspect, the invention relates to an animal cell with a modified genome that comprises a gene encoding a transcriptional regulator protein inserted into a first genetic safe harbour site; and an inducible cassette sing a c sequence operably linked to an inducible promoter inserted into a second genetic safe harbour site; n said inducible promoter is regulated by the transcriptional regulator protein and said first and second c safe harbour sites are different.

In a fifth aspect, the invention relates to ex vivo or in vitro use of a cell according to the third aspect or the fourth aspect for tissue engineering, optionally for the production of cultured meat.

In a sixth aspect, the invention relates to an ex vivo method for reducing the transcription and/or translation of an endogenous gene in an animal cell, comprising the following steps: a) targeted insertion of a gene encoding a transcriptional tor protein into a first genetic safe r site; and b) targeted insertion of an inducible cassette comprising DNA encoding a ding RNA sequence operably linked to an inducible promoter into a second genetic safe harbour site; wherein said promoter is regulated by the transcriptional regulator protein; wherein said non-coding RNA sequence suppresses the transcription or ation of an endogenous gene; wherein said first and second c safe harbour sites are different. 2b followed by page 3 In a seventh aspect, the invention relates to an ex vivo method for knocking out an endogenous gene in an animal cell, comprising the ing steps: a) targeted insertion of a gene encoding a transcriptional regulator protein and a gene encoding Cas9 into a first genetic safe harbour site; and b) ed insertion of an inducible te comprising a guide RNA operably linked to an inducible promoter into a second genetic safe r site; wherein said promoter is regulated by the transcriptional regulator protein; wherein said gRNA sequence targets the endogenous gene; and wherein said first and second c safe harbour sites are different.

The present invention thus relates to a method for controlling the transcription of a genetic sequence in a cell, comprising the following steps: a) targeted insertion of a gene encoding a transcriptional regulator protein into a first genetic safe harbour site; and b) targeted ion of an inducible te into a second genetic safe harbour site, wherein said inducible cassette ses said genetic sequence operably linked to an inducible promoter and said promoter is regulated by the transcriptional regulator protein; wherein said first and second genetic safe harbour sites are different.

Inducible cassette integration specifically into genomic safe harbour sites (GSHs) is preferred over random insertion into the genome. GSHs have been defined previously as ”intragenic or extragenic regions of the human genome that are able to accommodate the predictable expression of newly ated DNA without e effects on the host cell or organism. A useful safe harbour must permit sufficient transcription of the ed genetic sequence to yield desired levels of the protein (via further translation) or ding RNA. A GSH also must not predispose cells to malignant transformation nor alter cellular functions” (Sadelain et at, 2012, Nature Reviews Cancer, 12(1), 51—8. doi:10.1038/nrc3179).

The first genetic safe harbour site is utilised to introduce a gene encoding at least a transcriptional regulator protein. A transcriptional regulator protein (or transcription factor) increases gene ription of a gene.

Most transcriptional regulators are DNA-binding proteins that bind to ers or promoter-proximal elements operably linked to the gene.

In some aspects, the riptional regulator protein is constitutively expressed, and is permanently expressed in a cell. The transcriptional regulator n may thus be operably linked to a constitutive promoter. Constitutive promoters direct gene expression uniformly in most tissues and cells at all stages of growth and development. Constitutive promoters confer high levels of gene expression when used in the methods of the present invention.

Further genetic material including genes may be inserted into the first GSH with the transcriptional regulator protein. Such genes may include one or more markers such as green fluorescent protein (GFP) which can be used to show, for example, that the transcriptional regulator protein has been successfully inserted. Other options include genes that allow gene g, for example Ca59 and derivatives or CasL and tives, and reporter sequences that can be used to assay endogenous or ous expression of specific genes in the cell.

The second GSH is utilised to introduce an ble cassette in which the desired genetic sequence is operably linked to an inducible promoter. Such a promoter enables transcription only when correctly induced by the transcriptional regulator n. The transcriptional regulator protein may be controlled by a substance which is exogenously supplied to the cell. Thus, the presence of the exogenous substance may permit or block expression from the inducible promoter. An example of such controllable expression is the Tet-ON system which is described further herein.

Further inducible cassette(s) may be inserted into further GSHs, said GSHs are distinct from the first and second GSH mentioned above.

One or more genetic ces may be controllably transcribed from within the second and/or further GSH.

Indeed, the inducible cassette may contain 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 genetic sequences which it is desired to insert into the GSH and the transcription of which be controllably induced.

The genetic sequence or sequences which it is desired to insert into the GSH or GSHs are present within the inducible cassette, ly linked to an inducible promoter. These genetic sequences can be any suitable sequence, which are capable of being ribed into RNA once the activity of the promoter has been induced. Suitable c sequences include but are not limited to transgenes (protein coding genes, in which the RNA produced is messenger RNA (mRNA) is translated into a ptide), non-coding RNA (ncRNA — including but not limited to shRNA, antisense RNA (asRNA), guide RNA (gRNA), microRNA (miRNA), small interfering RNA (siRNA), trans-acting RNA (tasiRNA), antagomirs, rs, miRNA sponges, and any other functional RNA).

The inducible tes may e additional genetic material to be inserted into the second or r GSH.

Such additional genetic material may include one or markers such as green fluorescent protein (GFP) to indicate that the transcription is occurring. Alternatively, or additionally, genes such as antibiotic or drug resistance genes may allow for ion of successfully inserted inducible tes. Moreover, the inducible expression of a particular gene to study its function or of sequences that will interfere with its function may be desirable. Equally, expression of genes to e or obstruct ical functions of the cell or influencing cells in other part of the sm may be desirable, including the expression of growth s, peptide hormones, including insulin etc.

Technically, the insertions into the first and/or second GSH may occur on one chromosome, or on both chromosomes. The GSH exists at the same genetic loci on both chromosomes of diploid organisms. Insertion within both chromosomes is advantageous since it may enable an increase in the level of transcription from the inserted genetic material within the inducible cassette, thus achieving particularly high levels of ription.

The insertions into the GSHs may be controlled Specific insertion of genetic material into the particular GSH based upon customised site-specific generation of DNA double-strand breaks (DSB) at the GSH may be achieved. The genetic material may then be introduced using any suitable mechanism, such as homologous recombination. Any method of making a specific DSB in the genome may be used, but preferred systems include CRISPR/Ca59 and modified versions thereof, ZFNs and the TALEN system.

Furthermore, the insertion of the transcriptional regulator and/or inducible cassette can be designed to be reversible and the inserted genetic material may be removed and/or replaced with and alternative transcriptional regulator/inducible cassette as appropriate. Methods of replacing the transcriptional regulator and/or inducible te form part of the ion. Such ement may be useful where a culture of cells has been modified successfully with one transcriptional regulator and/or one inducible te, and it is desirable to replace the transcriptional regulator and/or inducible cassette. This takes advantage of the already successful insertion and may allow for larger ions to be made. In order to m this aspect of the invention, the insertions may include cleavable sequences to allow for the removal of all or part of the insertion from the GSH, such as a portion of the insertion. Preferred methods of removal or replacement include recombinational approaches.

Further, the invention relates to the vectors suitable for insertion of the transcriptional tor and/or inducible cassette into the GSH.

In one , the present invention provides a method for controlling the expression of a transgene in a cell, comprising the following steps: a) ed insertion of a gene encoding a transcriptional regulator protein into a first genetic safe harbour site; and b) targeted ion of a transgene operably linked to an inducible promoter into a second genetic safe r site, wherein said inducible promoter is regulated by the transcriptional regulator protein; wherein said first and second genetic safe harbour sites are different.

In this aspect of the invention, the inducible cassette described previously comprises a transgene ly linked to an inducible promoter. In this aspect of the invention, the desired genetic sequence included within the inducible cassette is a transgene, preferably a protein-encoding gene. Thus, the transcription and translation (expression) of the ene may be lled within the cell. The age of the present method is that it permits overexpression of the ene, if required.

Further, in this aspect of the invention, a further cal or different ene may be inserted into a further GSH, which is different to the first and second GSH. Such a transgene is operably linked to an inducible promoter as described above.

In one aspect, the t invention provides a method for controlling the transcription of a non—coding RNA in a cell, comprising the following steps: a) targeted insertion of a gene encoding a transcriptional regulator protein into a first genetic safe harbour site; and b) targeted insertion of an inducible cassette into a second genetic safe harbour site, wherein said inducible cassette comprises DNA encoding a non-coding RNA sequence operably linked to an inducible promoter and said promoter is regulated by the transcriptional regulator protein; wherein said first and second genetic safe r sites are different.

Further, in this aspect of the invention, a further identical or different inducible cassette may be inserted into a further GSH, which is different to the first and second GSH. Such an inducible cassette may comprise DNA encoding a non-coding RNA sequence or any other genetic sequence operably linked to an inducible promoter and said promoter is regulated by the riptional regulator protein.

More particularly, this method allows for the knockdown of an endogenous gene in the cell. Thus, the present invention provides a method for reducing the ription and/or translation of an endogenous gene in a cell, comprising the following steps: a) targeted insertion of a gene encoding a transcriptional regulator protein into a first genetic safe harbour site; and b) targeted insertion of an ble cassette into a second genetic safe harbour site, wherein said inducible te comprises DNA encoding a non—coding RNA sequence operably linked to an inducible er and said promoter is regulated by the transcriptional regulator protein and wherein said non-coding RNA sequence suppresses the transcription or ation of an endogenous gene; wherein said first and second genetic safe harbour sites are different.

Further, in this aspect of the invention, a further identical or different inducible te may be inserted into a further GSH, which is different to the first and second GSH. Such an inducible cassette may comprise DNA encoding a non-coding RNA sequence or any other genetic sequence operably linked to an ble promoter and said promoter is regulated by the transcriptional regulator protein.

In any aspect or embodiment, the endogenous gene may encode a protein or a non-coding RNA.

In the above two aspects of the invention, the inducible cassette(s) comprises a DNA encoding a non-coding RNA, i.e. an RNA which is onal but is not translated into protein. This ding RNA may be any suitable RNA, such as those discussed previously, but is preferably short n RNA (shRNA). In the latter aspect of the invention, the non-coding RNA may effect gene knockdown in any suitable way, by blocking gene transcription or translation or preventing expression in general. Ultimately, the expression of said gene is reduced or blocked, but the gene itself remains intact.

Alternatively, the non-coding RNA comprised within the sequence of the inducible cassette may e RNAs which can be used to knockout an nous gene in a cell, notably to replace or disrupt the gene itself. le non-coding RNAs that could be used for this aspect of the ion e elements of the CRISPR/Cas9 platform, more particularly the guide RNAs (gRNA) that are directed to target the endogenous gene.

Thus, in one aspect, the present invention provides a method for knocking out of an endogenous gene in a cell, comprising the following steps: a) targeted insertion of a gene encoding a transcriptional regulator protein and a gene encoding Ca59 or a derivative thereof into a first genetic safe harbour site; and b) targeted insertion of an inducible cassette into a second genetic safe harbour site, wherein said inducible cassette comprises a guide RNA operably linked to an inducible promoter and said promoter is regulated by the transcriptional regulator protein and wherein said gRNA sequence targets the endogenous gene; wherein said first and second c safe harbour sites are different.

Further, in this aspect of the invention, a further identical or different inducible cassette may be inserted into a r GSH, which is different to the first and second GSH. Such an inducible cassette may comprise any genetic sequence operably linked to an ble promoter and said promoter is regulated by the transcriptional regulator protein.

Thus, in the above aspect of the invention, the transcription of the gRNA is controllably induced.

In a further aspect, the present invention provides a method for reducing the transcription and/or ation of an endogenous gene in a cell, sing the following steps: a) targeted insertion of a gene encoding a transcriptional tor n into a first allele of a genetic safe harbour site; and b) targeted insertion of an inducible cassette into a second allele of the same genetic safe harbour site, wherein said inducible cassette comprises DNA encoding a ding RNA sequence operably linked to an inducible promoter and said promoter is ted by the transcriptional regulator protein and n said non-coding RNA sequence suppresses the transcription or translation of an endogenous gene.

Further, the t invention provides a method for knocking out of an endogenous gene in a cell, comprising the following steps: a) targeted insertion of a gene encoding a transcriptional regulator protein and a gene encoding Ca59 or a derivative thereof into a first allele of a genetic safe harbour site; and b) ed insertion of an inducible cassette into a second allele of the same genetic safe harbour site, wherein said inducible cassette comprises a guide RNA operably linked to an inducible promoter and said promoter is regulated by the riptional tor protein and wherein said gRNA sequence s the endogenous gene.

Such single-step knock—outs or knock downs are new and may form part of the invention.

In one aspect, the present invention provides a method for the forward mming of pluripotent stem cells, comprising the steps of: a) targeted insertion of a gene encoding a transcriptional regulator protein into a first genetic safe harbour site; and b) targeted insertion of an inducible cassette into a second genetic safe harbour site, n said inducible cassette comprises a genetic sequence encoding a key lineage transcription factor operably linked to an inducible promoter, said inducible promoter is regulated by the transcriptional regulator protein; and wherein said first and second genetic safe harbour sites are ent. r or additional inducible cassette(s) may be inserted into further GSHs distinct from the first and second The forward programming of pluripotent stem cells into particular mature cell types is highly desirable and can be achieved using the dual-targeting platform of the present invention. Particular methods for n cell types are bed below.

In one , the t invention provides a method for the production of myocytes from pluripotent stem cells, comprising the steps of: a) targeted insertion of a gene encoding a transcriptional regulator protein into a first genetic safe harbour site; and b) targeted insertion of the MYODl gene operably linked to an inducible promoter into a second genetic safe harbour site, wherein said inducible promoter is regulated by the transcriptional regulator protein; wherein said first and second genetic safe harbour sites are different, and culturing said cells in the presence of retinoic acid.

The MYODl gene is the gene encoding the Myogenic Differentiation 1 protein. Preferably, the retinoic acid (RA) is all-trans RA.

In another aspect, the present invention provides a method for the production of myocytes from pluripotent stem cells sing MYODl, sing culturing said cells in the presence of retinoic acid.

Preferably, the RA is ans RA. Preferably, the cells are overexpressing MYODl.

In a further , the present invention provides a method for the production of oligodendrocytes from pluripotent stem cells, comprising the steps of: a) targeted ion of a gene encoding a transcriptional tor protein into a first genetic safe harbour site; and b) targeted insertion of any combination of the 50X 10, OLIGZ, NKX2.2, AND NKX6.2 genes ly linked to an inducible promoter into a second genetic safe harbour site, wherein said inducible promoter is regulated by the transcriptional regulator protein; wherein said first and second genetic safe harbour sites are different, The $0X—10, OLIGZ, , NKX6.2 genes encode the ription factor SOX—lO, OLIGZ, NKX2.2, AND , respectively. ption of Figures Figure 1 (a — d): Validation of an optimized dual genomic safe harbor targeted overexpression system. Figure 1(a) Design of the gene targeting vectors for the 6 and AAVSl loci. HAR: homology arm, SA: splice acceptor, T2A: T2A ribosomal skipping signal; Neo: neomycin resistance gene; Puro: puromycin resistance gene. pA: polyadenylation signal; CAG: constitutively active CAG promoter; rtTA; third generation rtTA; TRE: inducible Tet-Responsive Element; EGFP: enhanced green fluorescent protein. Figure 1(b) shows EGFP ion and rescue kinetics (1(c)) in EGFP expressing hESCs detected by flow cytometry (median fluorescence intensity, MFI). Results are from two biological replicates per time point and are expressed as mean i SEM. All values were normalized to the maximum fluorescence ity after 5 days of cline (referred to as day 0 in the figure). Figure 1(d) shows Doxycycline dose-response for EGFP overexpression in EGFP expressing hESCs following induction with doxycycline for 5 days. Results are from two biological replicates per condition, and are expressed as mean i SEM. All values were normalized to the maximum fluorescence intensity measured in the experiment. EGFP expression levels in rgeted constitutive CAG— EGFP hPSCs and in dual GSH-targeted inducible TRE-EGFP hPSCs following induction with doxycycline. Wild- type hPSCs and non-induced TRE—EGFP cells were included as negative controls.

Figure 2 (a — d): Overview of the experimental ch and results for rapid single step conversion of hPSCs into neuronal cells (i-Neurons) following doxycycline (dox) treatment. Figure 2(a) is a schematic of this sion, in which cells transformed according to the invention with NGN2 are induced to differentiate into neuronal cells following Dox treatment. Figure 2(b) demonstrates the forward programming time course of i- Neuron generation from hESCs documented by quantitative RT-PCR-analysis, which demonstrates the al expression pattern of pan-neuronal (MAP2, SYP), forebrain (BRNZ, FOXG 1) and glutamatergic neuronal marker genes (VG LUTZ, GRIA4). Cells were analyzed at the indicated days of doxycycline treatment.

Values are shown relative to the nous housekeeping gene PBGD and normalized to pluripotency conditions. Results are from three biological ates per time point and are expressed as mean i SEM.

Figure 2(c) depicts the quantification of Blll-tubulin (TU BB3) positive neuronal cells by immunostaining in i- Neurons derived from hESCs after one week of induction. Undifferentiated cells were used as negative control (Control), and numbers are reported for i—Neuron generation in newly isolated NGNZ sing hESCs and after 25 passages (+P25). Figure 2(d) are cell photographs depicting the d programming time course of i- Neuron generation from hESCs via serial phase contrast images which illustrate morphological changes.

Figure 3 (a-d): Forward programming of hPSCs into skeletal myocytes. Figure 3a shows a schematic of the rapid single step conversion of hPSCs into skeletal myocytes by ble overexpression of MYODl and treatment with retinoic acid. Figure 3b shows quantitative -analysis of the temporal expression pattern of myocyte marker genes during i-Myocyte generation from hESCs. All values are shown relative to the hPSCs.

Results are from three ical replicates per time point and are expressed as mean 1 SEM. Figures 3 (c) and (d) show quantification of MHC positive cells by flow cytometry ten days after ion demonstrating that YODl hPSCs retain their myogenic potency even after extended e periods and passaging (p) following the targeted integration of the MYODl system. Undifferentiated cells were used as negative control (Control), and figures are ed for i-Myocytes generation in newly isolated OPTi-MYODl hESCs, or in the same cells following 50 passages (+P50).

Figure 4 (a — f): Targeting strategy for the dual GSH targeted Tet-ON overexpression system. Figure 4 (a) s the experimental workflow for the sequential targeting of the hROSA26 and AAVSl loci in hPSCs. Key: Cas9n: D10A nickase mutant Cas9 clease from S. Pyogenes; ZFN: inger nuclease; Neo: neomycin; Puro: puromycin; rtTA: third generation reverse-tetracycline Trans-Activator;. This s an ble EGFP expression system (i-EGFP) Figure 4(b) depicts a schematic of the hROSA26 targeting strategy. Figure 4(c) depicts the AAVSl targeting strategy. The key for figures 4(b) and (c): R26-prom: ROSA26 locus promoter (THUMPD3-ASl gene); AAV-prom: AAVSl locus promoter (PPPlRlZC gene); ZFN: zinc-finger nucleases; 5’— HAR/3’—HAR: upstream/downstream gy arm. SA: splice acceptor; T2A: T2A peptide; pA: polyadenylation signal; CAG: CMV early er, chicken B-actin and rabbit B-globin hybrid promoter; TRE: Tet-responsive element; EGFP: enhanced green fluorescent protein. Figure 4 (d) depicts the tic of the genotyping strategy used to identify correctly targeted hROSA26 and AAVSl targeted hPSC lines.; GSH-prom: GSH promoter (hROSA26 and AAVSl, respectively); WT: ype; Inducible te: entire exogenous sequence integrated following targeting. Locus PCR: PCR spanning the targeted locus with both primers binding exclusively to genomic DNA outside the genomic sequence corresponding to the homology arms. Note that due to its high GC-content the CAG promoter cannot be amplified by routine PCR. Therefore, correct insertion of the CAG-containing expression cassette results in loss of a PCR amplicon. The ce of the wild- type band indicates the presence of non-targeted alleles; loss of the wild-type band indicates homozygous targeting. 5’-|NT/3’-|NT: PCRs: PCRs spanning the 5'— and 3’-insertion site, respectively. Correctly sized PCR amplicons indicate correct integration. 3’BB PCR: PCR spanning the homology arm/targeting vector ne junction. The presence of a PCR product indicates non—specific off-target integration of the donor plasmid.

Figure 4(e) is a gel photograph which shows the genotyping s for selected hROSA26-CAG-rtTA targeted heterozygous (HET) and homozygous (HOM) H9 hESCs. Figure 4(f) is a gel photograph that shows the ping results for selected AAVSl-TRE-EGFP targeted heterozygous (HET) and gous (HOM) H9 hESCs. 1kb+: lkb plus DNA ladder; WT: wild—type hESCs; PL: targeting plasmid; H20: water control.

Figure 5 (a-e): Development of an optimized inducible overexpression platform (OPTi-OX) based on hPSC dual GSH targeting. Figure 5(a) shows a dual GSH-targeted ble EGFP H9 hESCs were pooled into four experimental groups depending whether one or both alleles of the hROSA26 and AAVSl loci, respectively, were successfully targeted. Figure 5(b) shows detection of the rtTA protein by Western blot in successfully targeted - and gous H9 hROSA26-CAG-rtTA hESCs. Human ESCs carrying a second generation rtTA in a random genomic position were ed as control sample. a-tubulin: loading control. Figure 5(c) depicts flow cytometry analysis for the representative examples of the various dual GSH—targeted inducible EGFP hESCs described in Figure 5(a). Figure 5(d) shows median fluorescent intensity (MFI) of EGFP expression in the various dual GSH—targeted inducible EGFP hESCs described in figure 5(a). Cells were ed by flow cytometry in control conditions (no doxycycline, CTR) or following 5 days of doxycycline treatment (DOX). Each data point represents an individual clonal line. CAG—EGFP hESCs and wild-type (WT) hESCs were ed for comparison. Statistical is of doxycycline-treated groups , as indicated) demonstrated that EGFP expression levels were highest in double-homozygous clones (One-way ANOVA with post-hoc Dunnet’s test; F (2, 10) = 25.34, p=0.0001; **** p<0.0001; ** p=0.0026). This condition was ed forfurther experiments.

Figure 5(e) shows the percentage of EGFP+ cells in the various dual GSH-targeted i-EGFP hESCs described in Figure 5(a).

Figure 6(a-d): Characterization of the OPTi—OX platform in hPSCs and during germ layer differentiation. Figure 6(a) depicts flow cytometry analysis of EGFP levels in successfully targeted live hPSCs and after their differentiation into the three germ layers treatment following treatment with doxycycline for five days. The acquisition settings were set to include the high levels of induced EGFP expression . The non-induced control populations (CTR) are located directly next to the left y—axis. Figures 6(b) and 6(c) show a summary of the flow cytometry plots in 6(a), including the median fluorescent ity (MFI) and the percentage of EGFP+ cells. Figure 6(d) shows a bar chart of quantitative RT—PCR results of EGFP mRNA expression levels of homozygous pluripotent stem cells and following differentiation into the three germ layers. WT: wild type; Figure 7: Characterization of human i-Neurons. Quantitative RT—PCR results demonstrate rapid downregulation of the pluripotency factors NANOG and OCT4 upon treatment with cline.

Figure 8: RA signaling during myocyte induction. This figure shows qPCR is of the six retinoid and retinoid receptors during myocyte induction demonstrates expression of RARu, RARB and all three RXR isoforms, but not of RARy throughout the course of i-myocyte induction. A is a, B is [3 and G is y.

Figure 9(a) to 9(c): terization of the development of OPTi-MYODl hESCs into human i-Myocytes. Figure 9(a) shows forward programming time course of OPTi-MYODl hPSCs into induced myocytes. logical changes were documented with ted phase contrast images that were acquired every 30min with a Nikon Biostation IM time lapse system. Scale bars: ZOOum. Figure 9(b) depicts qPCR results demonstrating rapid downregulation of the pluripotency factors NANOG and OCT4 upon treatment with doxycycline of OPTi- NGNZ hESCs (left graph). All five major human skeletal myocyte specific myocyte heavy chain isoforms (encoded by the MYH gene family) are strongly upregulated during myocyte d programming (right graph). These include the two isoforms that are expressed during embryonic and postnatal muscle pment (embryonic isoform MYH3; neonatal isoform MYH8) and three ms that are usually expressed in adult human skeletal muscle [MYH7 in slow-twitching (type |) fibers; MYHZ in witching fatigue-resistant (type Ila) fibers, and MYHl in fast—fatigable (type le) fibers]. In contrast, MYH4 which represents the constituting MHC—isoform in fast-twitching, fast-fatigable myocyte fibres in cats is not expressed in significant amounts in humans (<1%) and is also not induced throughout the forward programming time course. Figure 9(c) depicts induced skeletal myocytes express a broad range of typical marker proteins, including n lized through Iuor488—conjugated Phalloidin toxin), Neural Cell Adhesion molecule (NCAM), Desmin (DES), Myosin Heavy Chain (MYH), Titin (TTN), a-Actinin (ACTNZ) and Troponin T (TNNT), but not the myoblast itor markers PAX3 and PAX7. All s were counterstained with in (MYOG). Scale bars: 50pm. DAPI: nuclear staining.

Figure 10: These three graphs depict the qPCR results for total MYODl, endogenous MYODl, and MYOG 2 days post induction of OPTi-MYODl hPSCs with different concentrations of doxycycline. The qPCR results are shown 48h post induction with different concentrations of doxycycline. Expression is plotted relative to the endogenous housekeeping gene PBGD.

Figure 11: A depiction of the Tet—ON system. The Tet-ON consists of two components: At the top the activator cassette is depicted, in which a tutive promoter (cP) drives expression of rtTA (reverse-tetracycline Trans—Activator). RtTA is a fusion protein that consists of a mutant form of the prokaryotic Tet Repressor (TetR) and the transcriptional trans-activator domain VP16 (derived from herpes simplex virus). At the bottom the responder domain is depicted. It consists of an inducible promoter (TRE, Tet Responsive Element) and the gene of interest. The TRE is an artificial er responsive to rtTA. It consists of 7 serial tet operons (tetO7) and a strong minimal CMV promoter , which itself is not active and only recruits the transcriptional ery upon binding of rtTA to the seven tet operons. Doxycycline, a tetracycline tive, is required for binding of the mutant TetR to the TRE, leading to expression of the inducible cassette, in this case EGFP. (pA: polyadenylation signal).

Figure 12 (a-d) Forward programming of hPSCs into oligodendrocytes. Figure 12(a) depicts a tic of the experimental approach for rapid conversion of OPTi-OLIGZ-SOXlO hPSCs into oligodendrocyte lineage cells (i- OPCs and i-OLs). Figure 12(b) shows the quantification of BrdU-positive cells following 3 serial passages every 4 days and concomitant BrdU-pulses each g 4 days (P = passage number). Figure 12(c) shows quantitative RT-PCR-analysis of the temporal expression pattern of genes encoding for the myelin associated proteins (CNP, MAG, MBP, M06, and PLP) during i-Oligodendrocyte generation from hPSCs. OPTi-OLIGZ-SOXlO hPSCs were induced in oligodendrocyte media supplemented with PDGFaa and FGF2. After one week of induction mitogens were withdrawn to enable terminal differentiation. All values are shown relative to the endogenous housekeeping gene PBGD and ized to pluripotency conditions. Results are from 2-3 biological ates per time point and are expressed as mean i SEM. Figure 12(d) depicts the quantification of CNP and PLP positive cells by immunostainings in i-oligodendrocytes d from OPTi-OLIGZ-SOXlO hPSCs after 20 days of induction. Undifferentiated cells were used as negative control, and figures are reported for i- endrocytes in newly ed OPTi-NGN2 hPSCs and after 50 passages (+P50).

Figure 13 is a schematic representation of the principles of the present invention. Essentially, this depicts the insertion into two different genetic safe harbor sites at the core of the present invention. One insertion controls the expression of the genetic sequence within the inducible cassette in a second insertion. Additional genetic material can be included in polycistronic vector constructs as shown. Further, more than two c safe harbor sites may be targeted, such that multiple inducible tes or other genetic material may be placed under the control of the modulator placed in the first GSH site.

Figure 14 (a to f) are depictions of the results showing the development of an inducible knockdown system based on dual GSH targeting of hSPCs. Figure 14a shows the experimental approach — H1 — H1 promoter, TO — tet operon, tetR — tetracycline repressor. Figure 14b is a schematic of the transgenic alleles generated to obtain hESCs expressing an EFGP reporter transgene that could be silenced using an inducible EGFP shRNA. Figure 14c shows EGFP expression in the absence or ce of tetracycline for 5 days in hESCs targeted with the indicated ations of inducible EGFP shRNA and tetR (STD = wild type standard, OPT = codon optimized). Double-targeted hESCs that did not carry the EGFP shRNA were used as negative controls. n.s.=p>0.05 (non-significant), **=p>0.01, ***=p>0.001 VS same tetR line no tet and no shRNA. Figure 14d is a representative n blot for tetR in ROSA26-targeted hESCs expressing STD or OPT tetR. HET= heterozygous targeting, HOM= homozygous targeting. hESCs with STD tetR random integration are shown as a ve reference, while WT h9 hESCs are negative controls. TU B4A4A is a loading control. Various protein amounts were loaded to facilitate tative comparison. Figure 14 (E): EGFP knockdown and rescue kinetics in EGFP OPTiKD hESCs measured by flow cytometry (MFI) and qPCR (mRNA).

Results are from 2 independent cultures per time point. Figure 14(F): Tetracycline dose—response curve for EGFP own in EGFP OPTiKD hESCs. The half-maximal inhibitory concentration (ICSO) is reported. Results are from 2 independent cultures per dose, and the mean is shown.

Figure 15 (a, b and c) Validation of the ROSA26 and AAVSl loci as bona fide GSH Figure 15a shows the experimental ch behind the generation of GSH EGFP er hPSCs to test GSH expression during entiation. Neurons, oligodendrocytes, and astrocytes were ed in bulk cultures containing a mixture of these cell lineages, while all other cell types were individually generated. Figure 15b is a schematic of the ROSA26 and AAVSl EGFP reporter transgenic alleles. R26-prom: ROSA26 locus promoter; AAV-prom: AAVSl locus promoter; 5’- HAR/3’-HAR: upstream/downstream homology arm; SA: splice acceptor; T2A: self-cleaving T2A peptide; Neo: neomycin resistance; Puro: puromycin resistance; pA: polyadenylation signal; CAG: CAG promoter; EGFP: enhanced green fluorescent protein. Figure 15 (C): EGFP expression in absence or presence of tetracycline for 5 days in hESCs ed with the indicated combinations of inducible EGFP shRNA and tetR (wild-type standard tetR, STDtetR, or optimized tetR, OPTtetR). Double-targeted hESCs that did not carry the EGFP shRNA were used as negative controls. Results are from 2-3 individual lines per condition (table 1). n.s.=p>0.05 (non-significant), **=p<0.01,***=p<0.001 VS same tetR line no tet and no shRNA (ANOVA with oc Holm-Sidak comparisons).

Figure 16 (a-d) Generation of ROSA26 and AAVS1 EGFP reporter hESCs. Fig 16(A): Schematic of the ROSA26 targeting approach and of the genotyping strategies used to identify correctly targeted lines. Ca59n: D10A nickase mutant Ca59 endonuclease from S. Pyogenes. R26-prom: ROSA26 locus promoter D3-ASl gene); 5’-HAR/3’-HAR: upstream/downstream homology arm; ene: region integrated following gene targeting; Locus PCR: PCR product of ype ROSA26 locus (indicating a non—targeted allele); Locus PCR/Loss-of—allele: PCR product of targeted allele/PCR that fails if the transgene contains the h CAG promoter ative of expected transgene targeting); 5’ INT/3’ INT PCR: PCR product of transgene 5’-end/3’- end integration region ative of expected transgene targeting); 5’ BB/3’ BB PCR: PCR product of vector backbone 5’-end/3’-end (indicative of non—specific off-target plasmid integration). Note that similar ing and genotyping strategies were applied for the AAVSl locus targeting. Fig 16(B): Schematic of the ROSA26 transgenic alleles generated to test the best strategy for constitutive EGFP ced green fluorescent protein) expression. GFP: EGFP driven by the endogenous ROSA26 promoter (R26—prom; targeting vector pR26-Puro_ENDO-EGFP); EFla—EGFP: EGFP driven by the elongation factor it: er (targeting vector pR26—Neo_EFla—EGFP); CAG-EGFP: EGFP driven by the CAG promoter (targeting vector pR26-Neo_CAG- EGFP); SA: splice acceptor; Puro: cin resistance (puromycin N—acetyltransferase); Neo: neomycin resistance cin phosphotransferase II); pA: polyadenylation signal. Fig 16(C): Flow try quantification of the percentage of EGFP positive cells (EGFP+,- the gate is shown), and of the EGFP median fluorescence intensity (MFI) in representative ROSA26-EGFP reporter hESC clonal lines, or wild-type H9 hESCs.

Fig 16(D): Percentage of EGFP positive cells in ROSA26-EGFP reporter hESCs. Results are for 3 clones with heterozygous ROSA26 targeting per condition.

Figure 17. Validation of the optimized ble knockdown platforms ing hPSC differentiation. The plot shows EGFP expression measured by qPCR in absence (CTR) or presence of tetracycline for 5 days (TET) in the indicated cell types derived from EGFP OPTiKD (iKD) and sOPTiKD (siKD) hESCs. EGFP levels are reported ve to control conditions in the same line for each individual lineage. Abbreviations indicate the lineages described in Fig. 15 (pluri: undifferentiated). Results are from two independent cultures per condition.

Figure 18 (a — d). Development of an optimized ble CRISPR/Cas9 knockout platform in hPSCs. Figure 18a shows the experimental approach for the generation of inducible knockout (iKO) hPSCs. Figure 18b depicts a schematic of the cloning procedure to generate AAVSl targeting vectors with an inducible gRNA cassette.

Figure 18c shows the transgenic alleles generated to obtain hESCs expressing an EGFPd2 reporter transgene that could be knocked out by CRISPR/Cas9 using an inducible EGFP gRNA (EGFP sOPTiKO hESCs). Bsd: blasticidin resistance; EGFPd2: destabilized EGFP. Fig 18 (d): Flow cytometry quantification of EGFPd2 inducible knockout kinetics in O cells from figure 19c (gRNA 2 —TO) and b (gRNA 3 — 2T0). The percentage of EGFP positive cells was monitored daily following addition of tetracycline. Results are from 2 independent cultures.

Figure 19 (a to e): Development of an optimized inducible CRISPR/Cas9 knockout platform in hESCs. (A—D) depict representative flow cytometry for EGFPd2 sion in EGFPd2 homozygous sOPTiKO hESCs carrying the indicated combinations of gRNA (2 or 3) and inducible er (T0 or 2T0, see fig 19 e). Targeting vectors: uro_siKOEGFP-2 (19a), pAAV-Puro_siKO-2TO-EGFP-2 (19b), uro_siKO-EGFP-3 (19c), pAAV-Puro_siKO-2TO-EGFP-3 (19d). Cells were cultured in presence of tetracycline (TET) for 5 days, or maintained in control (CTR) conditions in the absence of tetracycline. Note that the histograms have been normalized so that the area under the curve equals to 1 (100%) for all samples presented, in order to facilitate direct visual comparison. Fig 19(e): Nucleotide sequences of inducible H1 Pol III ers for the sOPTiKO system ning one or two tet operons(H1-TO and H1-2TO, respectively). Key sequence features are highlighted. The restriction enzyme cut sites used for gRNA cloning are shown (Fig. 188). DSE: distal ce t; PSE: proximal sequence element; TETOZ: tet operon; +1: start position of RNA transcription. s 20 to 33 are depictions of the maps of various plasmids used within the Examples of the present application. These are: ) pSpCa59n(BB)_R26-R 21) pSpCas9n(BB)_R26—L 22) pR26_CAG_EGFP 23) pR26_CAG_rtTA 24) pZFN-AAVSl-L—ELD (zinc finger nuclease left) 25) pZFN—AAVSl-R—KKR (zinc finger nuclease right) 26) pAAV_CAG_EGFP (donor) 27) pR26—Neo_CAG-OPTtetR (hROSA26 targeting of codon-optimized tetR) 28) pAAV—Puro_iKD (AAVSl targeting of inducible shRNA) 29) pAAV-Neo_CAG-Cas9 (AAVSl targeting of Cas9) 30) pAAV-Puro_siKO (AAVSl targeting of inducible gRNA,) 31) pAAV—Puro_siKO—2TO (AAVSl targeting of inducible gRNA, version with 2 tet operons in promoter) 32) pAAV_TRE-EGFP (EGFP inducible pression, attached) 33) pAAV_TRE-MYOD1 (MYODl inducible overexpression for muscle) ed Description The inventors have ped a method that is useful for ble transcription of c sequences comprised within inducible cassettes in eukaryotic cells, and specifically pluripotent stem cells and their progeny.

It is particularly applicable to the forward programming of pluripotent stem cells, via overexpression of inducible cassettes within said stem cell that promote development of a particular mature cell type. Further, it is also applicable to the knockdown or ut of endogenous functions within the cell in order to study loss of function or alter cellular functions or behaviour in these cells. Knockdown or ut may apply to protein-encoding genes or to DNA sequences ng non—coding RNA. Either may be targeted by the methods of the present invention by knockout or knockdown. 3O This method is based upon the at least dual targeting of safe harbour sites in the genome of the stem cell, with the system for induced ription split over two or more GSH. However, this method is not limited to stem cells, and can be used to modify the genome of any cell type, for example in research or in gene therapy. In the s of the ion one GSH is modified to contain a riptional regulator that is required to induce transcription of the genetic sequence contained within the inducible cassette inserted into a different GSH elsewhere in the genome. The transcriptional regulator is preferably constitutively expressed. It is preferred that an exogenous substance/agent has to be supplied in order to control the activity of the transcriptional regulator protein and thus control expression of the inducible cassette. Since at least two separate GSH are used in the method of the invention, there are a total of four possible insertion loci, since each GSH exists on both chromosomes of a diploid organism. This increases the amount of transcription 40 possible from the cell if all four loci are modified using the method of the invention. An example of various outcomes of the targeted insertion is shown in figure 5a. Further, the method of the invention uses at least two different GSH sites. It will be understood that further GSH sites could be used to introduce further transcriptional regulators, inducible tes or any other genetic material including, but not limited to selectable markers, antibiotic or drug resistance genes, genes relating to the CRISPR/CasQ system or genes of n function.

Thus, the present invention relates to a method for controlling the expression of an inserted genetic sequence in a cell, sing the following steps: a) targeted ion of a genetic sequence encoding a transcriptional regulator protein into a first genetic safe harbour site; and b) targeted insertion of an inducible cassette into a second c safe harbour site, wherein said inducible cassette comprises said genetic sequence ly linked to an inducible promoter, and said promoter is regulated by the transcriptional regulator protein; wherein said first and second genetic safe harbour sites are different.

Further, in this aspect of the invention, a r identical or different inducible cassette may be inserted into a further GSH, which is different to the first and second GSH. Such an inducible cassette is as bed herein.

Insertions specifically within genetic safe harbour sites is preferred over random genome ation, since this is expected to be a safer modification of the genome, and is less likely to lead to unwanted side effects such as silencing natural gene expression or causing mutations that lead to cancerous cell types.

A genetic safe harbour (GSH) site is a locus within the genome wherein a gene or other genetic material may be inserted without any deleterious effects on the cell or on the inserted c material. Most beneficial is a GSH site in which expression of the inserted gene sequence is not perturbed by any read—through expression from neighbouring genes and sion of the inducible te minimizes interference with the endogenous ription programme. More formal criteria have been proposed that assist in the determination of whether a ular locus is a GSH site in future (Papapetrou et al, 2011, Nature Biotechnology, 29(1), 73—8. doi:10.1038/nbt.1717.) These criteria include a site that is (i) 50 kb or more from the 5’ end of any gene, (ii) 300 kb or more from any gene related to cancer, (iii) 300 kb or more from any microRNA (miRNA), (iv) located outside a transcription unit and (v) located outside ultraconserved regions (UCR). It may not be necessary to satisfy all of these proposed criteria, since GSH already identified do not fulfil all of the criteria. It is t that a suitable GSH will satisfy at least 2, 3, 4 or all of these criteria.

Further sites may be identified by looking for sites where viruses naturally integrate without disrupting natural gene expression.

Any suitable GSH site may be used in the method of the invention, on the basis that the site allows insertion of genetic material t deleterious effects to the cell and permits transcription of the inserted genetic material. Those skilled in the art may use this simplified criteria to identify a suitable GSH, and/or the more formal criteria set out above.

For the human genome, several GSH sites have been identified, and these include the AAVSl locus, the 6 locus and the CLYBL gene. The CCR5 gene and HPRT gene have also been mooted as possible GSHs, and further investigation may identify one or more of these as GSHs in the human genome.

The adeno-associated virus integration site 1 locus (AAVSl) is located within the protein phosphatase 1, regulatory subunit 12C (PPP1R12C) gene on human chromosome 19, which is expressed mly and ubiquitously in human tissues. This site serves as a specific integration locus for AAV serotype 2, and thus was identified as a possible GSH. AAVSl has been shown to be a favourable environment for transcription, since it comprises an open chromatin structure and native chromosomal tors that enable resistance of the inducible cassettes against silencing. There are no known adverse s on the cell ing from disruption of the PPP1R12C gene. Moreover, an ble cassette inserted into this site remains transcriptionally active in many diverse cell types. AAVSl is thus considered to be a GSH and has been widely utilized for targeted trangenesis in the human genome.

The hROSA26 site has been identified on the basis of sequence analogy with a GSH from mice (ROSA26 — reverse ed splice acceptor site #26). gh the orthologue site has been identified in humans, this site is not commonly used for inducible cassette insertion. The present inventors have developed a targeting system specifically for the hROSA26 site and thus were able to insert c material into this locus. The hROSA26 locus is on chromosome 3 (3p25.3), and can be found within the Ensembl database (GenBank:CR624523). The exact genomic co-ordinates of the integration site are 3:9396280-9396303: Ensembl. The integration site lies within the open reading frame (ORF) of the THUMPD3 long non-coding RNA (reverse ). Since the 6 site has an endogenous promoter, the inserted genetic material may take advantage of that endogenous promoter, or alternatively may be inserted operably linked to a promoter. lntron 2 of the Citrate Lyase ike (CLYBL) gene, on the long arm of Chromosome 13, was identified as a suitable GSH since it is one of the identified integration hot-spots of the phage derived phiC31 integrase.

Studies have demonstrated that randomly inserted inducible cassettes into this locus are stable and expressed.

It has been shown that insertion of inducible cassettes at this GSH do not perturb local gene expression bi et al, 2015, PLOS One, DO|:10.1371). CLYBL thus provides a GSH which may be suitable for use in the present invention.

CCR5, which is located on chromosome 3 (position 3p21.31) is a gene which codes for HIV-1 major co-receptor.

Interest in the use of this site as a GSH arises from the null mutation in this gene that appears to have no adverse effects, but predisposes to HIV-1 infection resistance. Zinc-finger nucleases that target the third exon have been developed, thus allowing for insertion of genetic material at this locus. Given that the l function of CCR5 has yet to be ated, the site remains a putative GSH which may have utility for the present invention.

The hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene encodes a transferase enzyme that plays a central role in the generation of purine nucleotides h the purine salvage pathway. Thus, further work is required to ensure insertions at this site do not disrupt normal cellular function. r, it has been mooted as a GSH site. Insertions at this site may be more applicable for mature cell types, such as modification for gene therapy.

GSH in other organisms have been identified and include ROSA26, HRPT and Hippll (H11) loci in mice.

Mammalian genomes may include GSH sites based upon pseudo attP sites. For such sites, hiC31 integrase, the Streptomyces phage-derived recombinase, has been developed as a non-viral insertion tool, because it has the ability to integrate a inducible cassette-containing plasmid carrying an attB site into pseudo attP sites.

GSH are also t in the genomes of , and modification of plant cells can form part of the present invention. GSH have been identified in the genomes of rice (Cantos et 0/, Front. Plant Sci., 26 June 2014, Volume 5, Article 302, http:f[dxdoi.org[10.3389/fgls.2014.00302).

In the methods of the ion, insertions occur at different GSH, thus at least two GSH are required for the method of the invention. The first GSH is modified by insertion of a transcriptional regulator protein. The second GSH is modified by the ion of an inducible cassette which ses a genetic sequence operably linked to an inducible promoter. Other genetic material may also be inserted with either or both of these elements. The c sequence operably linked to an inducible er within the inducible cassette is preferably a DNA sequence. The genetic sequence(s) of the inducible cassette preferably encode an RNA molecule, and are thus capable of being transcribed. The ription is controlled using the inducible promoter. The RNA molecule may be of any sequence, but is preferably a mRNA encoding a protein, a shRNA or a gRNA.

The first GSH can be any le GSH site. Optionally, it is a GSH with an endogenous promoter that is constitutively expressed; which will result in the inserted riptional regulator protein being constitutively expressed. A suitable GSH is the hROSA26 site for human cells. atively, the inserted transcriptional regulator protein is operably linked to a promoter, preferably a constitutive er. A constitutive promoter can be used in conjunction with an insertion in the hROSA26 site.

A transcriptional regulator protein is a protein that bind to DNA, preferably sequence—specifically to a DNA site located in or near a promoter, and either facilitating the binding of the ription machinery to the promoter, and thus transcription of the DNA sequence (a transcriptional activator) or blocks this s (a transcriptional repressor). Such entities are also known as transcription factors.

The DNA sequence that a transcriptional regulator protein binds to is called a transcription factor-binding site or response element, and these are found in or near the promoter of the regulated DNA sequence.

Transcriptional tor proteins bind to a response element and promote gene expression. Such proteins are preferred in the methods of the present invention for controlling inducible cassette expression. riptional repressor proteins bind to a response element and prevent gene expression.

Transcriptional regulator proteins may be activated or deactivated by a number of mechanisms including binding of a nce, interaction with other transcription s (e.g., homo— or hetero—dimerization) or coregulatory proteins, phosphorylation, and/or methylation. The transcriptional regulator may be controlled by activation or deactivation.

If the transcriptional regulator protein is a transcriptional activator protein, it is preferred that the riptional activator protein requires tion. This activation may be through any suitable means, but it is preferred that the transcriptional regulator protein is activated h the addition to the cell of an exogenous substance. The supply of an exogenous substance to the cell can be controlled, and thus the activation of the transcriptional regulator protein can be controlled. Alternatively, an exogenous substance can be supplied in order to deactivate a transcriptional regulator protein, and then supply withdrawn in order to activate the transcriptional regulator protein.

If the transcriptional regulator protein is a riptional repressor protein, it is preferred that the transcriptional repressor n requires deactivation. Thus, a substance is supplied to prevent the transcriptional sor protein repressing transcription, and thus transcription is permitted.

Any le transcriptional regulator n may be used, preferably one that is activatable or deactivatable.

It is preferred that an exogenous substance may be supplied to control the transcriptional regulator n.

Such transcriptional regulator proteins are also called inducible transcriptional regulator proteins. ycline-Controlled Transcriptional tion is a method of inducible gene expression where transcription is reversibly turned on or off in the ce of the antibiotic tetracycline or one of its derivatives (e.g. doxycycline which is more stable). In this system, the transcriptional activator protein is tetracycline — responsive transcriptional activator protein (rtTa) or a derivative thereof. The rtTA protein is able to bind to DNA at ic TetO operator sequences. Several repeats of such TetO sequences are placed upstream of a minimal promoter (such as the CMV promoter), which together form a tetracycline response t (TRE).

There are two forms of this system, depending on whether the addition of tetracycline or a derivative activates (Tet-On) or deactivates (Tet-Off) the rtTA protein.

In a Tet-Off system, tetracycline or a derivative thereof binds rtTA and deactivates the rtTA, rendering it ble of binding to TRE sequences, thereby preventing transcription of TRE—controlled genes. This system was first described in Bujard, et a] (1992). Proc. Natl. Acad. Sci. USA. 89 (12): 5547—51. 3O The Tet-On system is composed of two components; (1) the tutively expressed tetracycline — responsive transcriptional activator protein (rtTa) and the rtTa sensitive inducible promoter (Tet Responsive Element, TRE). This may be bound by ycline or its more stable derivatives, including doxycycline (dox), resulting in activation of rtTa, allowing it to bind to TRE sequences and inducing expression of ntrolled genes. The use of this may be preferred in the method of the invention. This system is depicted in Figure 11.

Thus, the transcriptional tor protein may thus be tetracycline—responsive riptional activator protein (rtTa) protein, which can be activated or deactivated by the antibiotic tetracycline or one of its derivatives, which are supplied exogenously. If the transcriptional regulator protein is rtTA, then the inducible promoter inserted into the second GSH site es the tetracycline response element (TRE). The exogenously supplied substance is the antibiotic tetracycline or one of its derivatives.

Variants and modified rtTa proteins may be used in the methods of the invention, these include Tet-On Advanced transactivator (also known as rtTAZS-MZ) and Tet—On 36 (also known as rtTA—V16, d from rtTAZS-SZ.

The ycline response element (TRE) generally consists of 7 repeats of the 19bp bacterial TetO sequence separated by spacer sequences, together with a minimal promoter. Variants and modifications of the TRE sequence are possible, since the minimal promoter can be any suitable promoter. Preferably the minimal promoter shows no or minimal expression levels in the absence of rtTa binding. The inducible promoter ed into the second GSH may thus comprise a TRE.

A modified system based upon tetracycline l is the T-RExTM System (Thermofisher Scientific), in which the transcriptional regulator protein is a transcriptional repressor protein, TetR. The components of this system include (i) an inducible promoter comprising a strong human cytomegalovirus immediate-early (CMV) promoter and two tetracycline operator 2 (TetOZ) sites, and a Tet repressor . The Tet02 ces consist of 2 copies of the 19 nucleotide sequence, S'-TCCCTATCAGTGATAGAGA-3' separated by a 2 base pair spacer. In the e of tetracycline, the Tet repressor forms a homodimer that binds with ely high affinity to each TetOZ sequence in the inducible promoter, and prevent ription from the er.

Once added, tetracycline binds with high affinity to each Tet repressor homodimer rendering it unable to bind to the Tet operator. The Tet repressor: tetracycline complex then dissociates from the Tet operator and allows induction of expression. In this instance, the transcriptional regulator n is TetR and the inducible promoter comprises two Tet02 sites. The exogenously supplied substance is tetracycline or a derivative thereof.

The invention further relates to a optimised tetR tR). This may be used in any method described herein, or for any additional use where inducible promotion is desirable. This entity was generated using mu|itparameter-optimisation of the ial tetR cDNA sequence. OPTtetR allows a ten-fold increase in the tetR expression when compared to the standard sequence (STDtetR). Homozygous R expression of tetR was sufficient to prevent shRNA leakiness whilst preserving knockdown induction in the es. The sequence for OPTtetR is included here, with the standard sequence shown as a ison. Sequences with at least 75%, 80%, 85% or 90% homology for this sequence are hereby claimed, more particularly 91, 92, 93, 94, 95, 96, 97 or 99% homology. Residues shown to be changed between STDtetR and OPTtetR have been indicated in the sequences, and it is preferred that these residues are not changed in any derivative of OPTtetR since these are t to be important for the improved properties. Any derivative would optionally retain these modifications at the indicated positions.

Other inducible expression systems are known and can be used in the method of the invention. These e the Complete Control Inducible system from Agilent Technologies. This is based upon the insect hormone ecdysone or its analogue ponasterone A (ponA) which can activate ription in mammalian cells which are transfected with both the gene for the Drosophi/a aster ecdysone receptor (EcR) and an inducible promoter comprising a binding site for the ecdysone receptor. The EcR is a member of the retinoid-X-receptor (RXR) family of nuclear receptors. In humans, EcR forms a heterodimer with RXR that binds to the ecdysone- responsive element (EcRE). In the absence of PonA, transcription is repressed by the heterodimer.

Thus, the transcriptional regulator protein can be a repressor protein, such as an ecdysone receptor or a derivative thereof. Examples of the latter include the VgEcR synthetic or from Agilent technologies which is a fusion of EcR, the DNA g domain of the glutocorticoid receptor and the transcriptional activation domain of Herpes Simplex Virus VP16. The inducible promoter ses the EcRE sequence or modified versions thereof together with a minimal promoter. Modified versions include the E/GRE recognition sequence of Agilent Technologies, in which mutations to the ce have been made. The E/GRE recognition sequence comprises ed half-site recognition elements for the retinoid-X-receptor (RXR) and GR binding domains. In all permutations, the exogenously supplied substance is ponasterone A, which s the repressive effect of EcR or derivatives thereof on the inducible promoter, and allows transcription to take place.

Alternatively, ble systems may be based on the synthetic steroid mifepristone as the exogenously ed substance. In this io, a hybrid transcriptional regulator n is inserted, which is based upon a DNA binding domain from the yeast GAL4 protein, a truncated ligand binding domain (LBD) from the human progesterone receptor and an activation domain (AD) from the human NF-KB. This hybrid transcriptional regulator protein is available from Thermofisher Scientific (Gene SwitchTM). Mifepristone activates the hybrid protein, and s transcription from the inducible promoter which comprises GAL4 upstream activating sequences (UAS) and the irus Elb TATA box. This system is bed in Wang, Y. et a/ (1994) Proc.

Natl. Acad. Sci. USA 91, 8180-8184.

The transcriptional regulator protein can thus be any suitable regulator protein, either an activator or repressor protein. Suitable transcriptional activator proteins are tetracycline — responsive transcriptional activator protein (rtTa) or the Gene Switch hybrid transcriptional regulator protein. Suitable repressor proteins include the Tet-Off version of rtTA, TetR or EcR. The transcriptional regulator proteins may be modified or derivatised as ed.

The inducible promoter can comprise elements which are suitable for binding or interacting with the transcriptional regulator protein. The interaction of the riptional regulator protein with the inducible promoter is preferably controlled by the exogenously ed nce.

The exogenously supplied substance can be any suitable substance that binds to or interacts with the transcriptional regulator protein. le substances include tetracycline, ponasterone A and mifepristone.

Thus, the insertion of the gene encoding a transcriptional regulator protein into the first GSH provides the control mechanism for the expression of the inducible cassette which is operably linked to the inducible promoter and inserted into a second, different, GSH site.

The transcriptional regulator protein gene may be ed for insertion with other genetic material. Such material includes genes for markers or reporter molecules, such as genes that induce visually identifiable characteristics including fluorescent and luminescent proteins. Examples include the gene that s jellyfish green fluorescent protein (GFP), which causes cells that express it to glow green under blue/UV light, luciferase, which catalyses a reaction with luciferin to produce light, and the red scent protein from the gene dsRed. Such markers or reporter genes are useful, since the ce of the reporter protein confirms protein expression from the first GSH, indicating sful ion. Selectable markers may r include resistance genes to antibiotics or other drugs. s or reporter gene sequences can also be introduced that enable studying the expression of endogenous (or ous genes). This includes Cas proteins, including CasL, Ca59 proteins that enable excision of genes of interest, as well as Cas-Fusion proteins that e changes in the expression of other genes, e.g. by acting as transcriptional enhancers or repressors. Moreover, non-inducible expression of lar tools may be desirable, including optogenetic tools, nuclear receptor fusion proteins, such as fen-inducible systems ERT, and designer receptors exclusively activated by designer drugs. Furthermore, sequences that code signalling factors that alter the function of the same cell or of neighbouring or even distant cells in an organism, including hormones autocrine or paracrine factors may be co-expressed from the same GSH as the transcriptional regulator protein.

Additionally, the further genetic al may include sequences coding for non-coding RNA, as discussed herein. Examples of such genetic material includes genes for miRNA, which may function as a genetic switch.

It is preferred that the gene encoding the transcriptional regulator protein is ly linked to a constitutive promoter. Alternatively, the first GSH can be selected such that it already has a constitutive er than can also drive expression of the transcriptional regulator n gene and any associated genetic al.

Constitutive promoters ensure sustained and high level gene expression. Commonly used constitutive promoters, including the human n promoter (ACTB), cytomegalovirus (CMV), elongation factor-la, (EFlot), phosphoglycerate kinase (PGK) and ubiquitinC (UbC). The CAG promoter is a strong synthetic promoter frequently used to drive high levels of gene expression and was constructed from the following sequences: (C) the cytomegalovirus (CMV) early enhancer element, (A) the promoter, the first exon and the first intron of chicken beta-actin gene, and (G) the splice acceptor of the rabbit beta-globin gene.

Further, the transcriptional regulator, plus any r genetic material may be provided together with cleavable sequences. Such sequences are sequences that are recognised by an entity capable of specifically cutting DNA, and include restriction sites, which are the target sequences for restriction enzymes or ces for ition by other DNA cleaving entities, such as nucleases, recombinases, ribozymes or artificial constructs. At least one cleavable sequence may be included, but preferably two or more are t. These cleavable sequences may be at any suitable point in the insertion, such that a selected portion of the insertion, or all of the insertion, can be selectively removed from the GSH. The method can thus extend to removal and/or replacement of the insertion or a portion thereof from the GSH. The cleavable sites may thus flank the part/all of the insertion that it may be desired to remove. The riptional regulator and/or the further genetic material may be removed using this method.

A portion of the insertion may be any part up to 99% of the insertion — i.e. 1-99%, 90%., 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or less than 10%.

It may be preferred that the portion of the insertion flanked by the ble sites includes the constitutive promoter. Alternatively, the constitutive promoter is not included in the portion flanked by the cleavable sequences.

A preferred ble sequence is the loxP site for Cre inase as it allows direct replacement of the removed insertion. Alternatively or additionally, the cleavable sequence is the rox site for Dre recombinase.

It is preferred that the insertion at the first GSH occurs at both loci in the genome, thus each allele is modified by insertion. This permits greater expression from the gene encoding the transcriptional regulator and any associated genetic material.

The second GSH can be any suitable GSH site. It may be preferred that the second GSH site is not associated with an endogenous promoter, such that the expression of the inserted inducible cassette is solely under control of the transcriptional regulator protein.

An inducible cassette includes a desired genetic sequence, preferably a DNA ce, that is to be transferred into a cell. The introduction of an inducible cassette into the genome has the potential to change the phenotype of that cell, either by addition of a genetic sequence that s gene expression or knockdown /knockout of endogenous expression. The methods of the invention provide for controllable transcription of the genetic ce(s) within the ble cassette in the cell.

The d genetic sequence for insertion is preferably a DNA sequence that encodes an RNA molecule. The RNA molecule may be of any sequence, but is preferably coding or non-coding RNA. Coding or messenger RNA codes for polypeptide sequences, and ription of such RNA leads to expression ofa protein within the cell. Non-coding RNA may be functional and may include without limitation: MicroRNA, Small interfering RNA, nteracting RNA, Antisense RNA, Small r RNA, Small lar RNA, Small Cajal Body RNA, Y RNA, Enhancer RNAs, Guide RNA, Ribozymes, Small hairpin RNA, Small temporal RNA, Trans—acting RNA, small interfering RNA and Subgenomic messenger RNA. Non-coding RNA may also be known as functional RNA. 3O Several types of RNA are regulatory in nature, and, for example, can downregulate gene sion by being complementary to a part of an mRNA or a gene's DNA. MicroRNAs (miRNA; 21—22 nucleotides) are found in eukaryotes and act through RNA interference , where an effector complex of miRNA and s can cleave complementary mRNA, block the mRNA from being translated, or accelerate its degradation. Another type of RNA, small interfering RNAs (siRNA; 20-25 nucleotides) act through RNA interference in a fashion similar to miRNAs. Some miRNAs and siRNAs can cause genes they target to be methylated, y decreasing or increasing transcription of those genes. Animals have Piwi-interacting RNAs (piRNA; 29-30 nucleotides) that are active in germline cells and are thought to be a defence against transposons. Many prokaryotes have CRISPR RNAs, a regulatory system similar to RNA interference, and such a system include guide RNA (gRNA). Antisense RNAs are widespread; most downregulate a gene, but a few are activators of transcription. Antisense RNA can act by binding to an mRNA, forming double—stranded RNA that is enzymatically degraded. There are many long noncoding RNAs that regulate genes in eukaryotes, one such RNA is Xist, which coats one X chromosome in female mammals and inactivates it. Thus, there are a multitude of functional RNAs that can be employed in the methods of the present invention.

Thus, the inducible cassette may include a genetic sequence that is a protein-coding gene. This gene may be not lly present in the cell, or may naturally occur in the cell, but controllable expression of that gene is ed. atively, the inducible cassette may be a mutated, ed or correct version of a gene present in the cell, particularly for gene therapy es or the derivation of disease models. The inducible cassette may thus e a transgene from a different organism of the same species (Le. a diseased/mutated version of a gene from a human, or a wild—type gene from a human) or be from a different species.

In any aspect or embodiment, the genetic sequence comprised within the ble cassette may be a synthetic sequence.

The inducible cassette may include any suitable genetic sequence that it is desired to insert into the genome of the cell. Therefore, the c ce may be a gene that codes for a protein product or a sequence that is transcribed into ribonucleic acid (RNA) which has a function (such as small nuclear RNA (snRNA), antisense RNA, micro RNA (miRNA), small interfering RNA (siRNA), er RNA (tRNA) and other non-coding RNAs (ncRNA), ing -RNA (chNA) and guide RNA (gRNA).

The ble cassette may thus include be any genetic sequence, the transcription of which it is desired to l within the cell. The genetic sequence chosen will be dependent upon the cell type and the use to which the cell will be put after modification, as discussed further below.

For e, for gene therapy methods, it may be desirable to provide the wild-type gene sequence as a component of the inducible cassette. In this scenario, the genetic sequence may be any human or animal protein-coding gene. Examples of protein-encoding genes include the human B-globin gene, human lipoprotein lipase (LPL) gene, Rab escort protein 1 in humans encoded by the CHM gene and many more.

Alternatively, the inducible te may express Growth factors, including BDNF, GDF, NG F, IG F, FGF and/or enzymes that can cleave pro-peptides to form active forms. Gene therapy may also be achieved by expression of an inducible cassette including a genetic sequence encoding an antisense RNA, a miRNA, a siRNA or any type of RNA that interferes with the expression of another gene within the cell.

Alternatively, should the cell be a stem cell, the inducible cassette may include a genetic sequence encoding a key lineage specific master regulator, abbreviated here are master regulator. Master regulators may be one or more of: transcription factors, transcriptional tors, cytokine receptors or ling molecules and the like. A master regulator is an expressed gene that influences the e of the cell expressing it. It may be that a network of master regulators is required for the lineage of a cell to be determined. As used herein, a master regulator gene that is expressed at the inception of a developmental e or cell type, participates in the specification of that lineage by regulating multiple downstream genes either directly or through a cascade of gene expression changes. If the master regulator is expressed it has the ability to re—specify the fate of cells destined to form other lineages. Examples of master regulators include the myogenic transcription factor MyoD and the hematopoietic transcription factor SCL. Particularly, master regulators include, but are not limited to: Neural lineages: Oligodendrocytes: SOXlO, OLIGZ, NKX2.2., NKX6.2; Astrocytes: NFIA, NFIB, and SOX9; Neurons: Ascll, neurogenin, and NeuroD , Pax6, 2, Ascll, Dlx2, and NeuroDl; Haematopoetic Cells , ing ocytes and Megakaryocytes: GATA1, FLll and TALl Mesenchymal lineages: Skeletal : MYOD; Cardiomyocytes: Gata4, Mef2c, Baf60c and Tbx5; Bone: L-Myc (RXOL) Runx2, Osterix, Oct4; Cartilage: c-Myc Klf4, SOX9; and Brown adipocytes: C/EBP—B and c-Myc Endoderm Pancreatic cell types:PDXl andGATA6.

Stem Cells: Epiblast SC: Oct4, Sox2, Klf4 and c-Myc Alternatively, or additionally, the genetic sequence or further genetic al may be genes whose function requires investigation, such that controllable sion can look at the effect of sion on the cell; the gene may include growth factors and/or cytokines in order for the cells to be used in cell transplantation; and/or or the gene may be components of a reporter assay.

Further, the genetic sequence may encode ding RNA whose function is to knockdown the expression of an endogenous gene or DNA sequence encoding non-coding RNA in the cell. Alternatively, the genetic sequence may encode guide RNA for the CRISPR—Cas9 system to effect endogenous gene knockout.

The methods of the invention thus extend to methods of knocking down endogenous gene expression within a cell. The methods are as described previously, and the inducible cassette comprises a genetic sequence ng a non—coding RNA operably linked to an inducible promoter, wherein the non—coding RNA suppresses the expression of said endogenous gene. The non—coding RNA may suppress gene expression by any suitable means including RNA interference and antisense RNA. Thus, the genetic sequence may encode a shRNA which can interfere with the ger RNA for the endogenous gene.

The reduction in endogenous gene sion may be partial or full — i.e. expression may be 50, 55, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% reduced compared to the cell prior to induction of the ription of the non-coding RNA.

The methods of the invention also extend to methods of knocking out nous genes within a cell, by virtue of the CRIPSR-Ca59 , gh any other suitable systems for gene knockout may be used. In this scenario, it is preferred that the Cas9 genes are constitutively expressed, and thus are included in the first GSH with the gene for the transcriptional regulator. Genetic sequences encoding the gRNAs may be included in the inducible cassette, which is inserted into the second GSH. gRNA is a short synthetic RNA composed of a scaffold sequence necessary for CasQ-binding and an imately 20 nucleotide targeting sequence which defines the genomic target to be modified. Thus, the genomic target of Ca59 can be changed by simply changing the targeting sequence present in the gRNA. Although the primary use of such a system is to design a gRNA to target an endogenous gene in order to knockout the gene, it can also be modified to electively activate or repress target genes, purify specific regions of DNA, and even image DNA. All possible uses are envisaged.

The inducible cassette es a genetic sequence operably linked to an ble promoter. A "promoter" is a nucleotide sequence which initiates and regulates transcription of a polynucleotide. An ”inducible promoter” is a nucleotide sequence where expression of a genetic sequence operably linked to the er is controlled by an e, co-factor, regulatory protein, etc. In the case of the present invention, the control is ed by the transcriptional regulator protein. It is intended that the term "promoter" or "control element" includes full-length promoter regions and functional (e.g., ls transcription or translation) ts of these regions. "Operably linked" refers to an arrangement of elements wherein the ents so described are configured so as to perform their usual on. Thus, a given promoter operably linked to a genetic sequence is capable of effecting the expression of that ce when the proper enzymes are present. The promoter need not be contiguous with the sequence, so long as it functions to direct the sion thereof. Thus, for example, intervening untranslated yet transcribed sequences can be t between the promoter sequence and the genetic sequence and the promoter sequence can still be considered "operably linked" to the genetic sequence. Thus, the term "operably linked" is intended to encompass any spacing or ation of the promoter element and the genetic sequence in the inducible cassette which allows for initiation of transcription of the inducible cassette upon recognition of the promoter element by a ription complex.

Further, other genetic material may also be operably linked to the inducible promoter. Further c material may include genes, coding ces for RNA, genetic material, such as markers or reporter genes.

Such additional genetic material has been discussed previously. In some circumstances, it may be desirable to include a suicide gene in the inducible cassette, should the genetic sequence itself not be a suicide gene for cancer gene therapy. The suicide gene may use the same inducible promoter within the inducible cassette, or it may be a separate inducible promoter to allow for separate control. Such a gene may be useful in gene therapy scenarios where it is desirable to be able to destroy donor/transfected cells if certain conditions are met. Suicide genes are genes that express a protein that causes the cell to undergo apoptosis, or alternatively may require an externally supplied tor or co-drug in order to work. The co-factor or co-drug may be converted by the product of the suicide gene into a highly cytotoxic entity.

Further, the inducible cassette may include ble sequences. Such sequences are sequences that are ised by an entity e of specifically cutting DNA, and include restriction sites, which are the target sequences for ction enzymes or sequences for recognition by other DNA ng entities, such as nucleases, recombinases, ribozymes or artificial constructs. At least one cleavable sequence may be included, but preferably two or more are t. These cleavable sequences may be at any suitable point in the cassette, such that a selected portion of the cassette, or the entire cassette, can be selectively removed from the GSH. The method can thus extend to removal and/or replacement of the cassette or a portion thereof from the GSH. The ble sites may thus flank the part/all of the genetic sequence that it may be desired to remove. The method may result in removal of the inducible cassette and/or the further genetic material.

A n of the cassette may be any part up to 99% of the cassette — i.e. 1-99%, 90%., 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or less than 10%.

It may be red that the portion of the insertion flanked by the ble sites includes the promoter operably linked to the genetic sequence. Alternatively, the promoter operably linked to the genetic sequence is not included in the portion flanked by the cleavable sequences.

A red cleavable ce is the loxP site for Cre recombinase as it allows direct ement of the removed insertion. Alternatively or additionally the cleavable site may be the rox site for Dre recombinase.

The transcriptional regulator n and the inducible cassette, together with any associated genetic material, are inserted into different GSH within the genome of the cell.

The insertions into the GSH are preferably specifically within the sequence of the GSH as described previously.

Any suitable technique for insertion of a polynucleotide into a specific sequence may be used, and several are described in the art. Suitable techniques include any method which introduces a break at the desired location and permits recombination of the vector into the gap. Thus, a crucial first step for targeted site-specific genomic modification is the creation of a double-strand DNA break (DSB) at the c locus to be modified.

Distinct cellular repair mechanisms can be exploited to repair the DSB and to introduce the desired sequence, and these are non-homologous end joining repair (NHEJ), which is more prone to error; and homologous recombination repair (HR) mediated by a donor DNA template, that can be used to insert inducible cassettes.

Several techniques exist to allow customized site-specific generation of DSB in the genome. Many of these 3O e the use of customized endonucleases, such as zinc finger nucleases (ZFNs), transcription activator—like effector nucleases (TALENs) or the clustered regularly interspaced short palindromic repeats/ CRISPR associated protein (CRISPR/CasQ) system (Gaj, T, et al ”ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering,” Trends Biotechnol, 31:397—405, July 2013).

Zinc finger nucleases are artificial enzymes which are generated by fusion of a zinc-finger DNA-binding domain to the nuclease domain of the restriction enzyme Fokl. The latter has a non-specific ge domain which must dimerise in order to cleave DNA. This means that two ZFN monomers are ed to allow dimerisation of the Fokl domains and to cleave the DNA. The DNA binding domain may be designed to target any genomic sequence of interest, is a tandem array of CyszHisz zinc fingers, each of which recognises three contiguous nucleotides in the target sequence. The two binding sites are separated by 5—7bp to allow optimal dimerisation of the Fokl domains. The enzyme thus is able to cleave DNA at a specific site, and target specificity is increased by ensuring that two al DNA—binding events must occur to e a double— strand break.

Transcription activator—like effector nucleases, or , are dimeric ription factor/nucleases. They are made by fusing a TAL or nding domain to a DNA cleavage domain (a nuclease). Transcription activator-like effectors (TALEs) can be engineered to bind practically any desired DNA sequence, so when combined with a nuclease, DNA can be cut at specific locations. TAL effectors are proteins that are secreted by Xanthomonas bacteria, the DNA binding domain of which contains a ed highly conserved 33—34 amino acid sequence with divergent 12th and 13th amino acids. These two positions are highly variable and show a strong correlation with specific nucleotide recognition. This straightforward relationship between amino acid sequence and DNA recognition has allowed for the engineering of specific DNA—binding domains by selecting a combination of repeat segments containing appropriate es at the two variable positions. TALENs are thus built from arrays of 33 to 35 amino acid modules, each of which targets a single nucleotide. By selecting the array of the modules, almost any sequence may be targeted. Again, the nuclease used may be Fokl or a derivative thereof.

Three types of CRISPR mechanisms have been fied, of which type II is the most studied. The CRISPR/Cas9 system (type II) utilises the Ca59 nuclease to make a double—stranded break in DNA at a site determined by a short guide RNA. The CRISPR/Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements. CRISPR are segments of prokaryotic DNA containing short repetitions of base sequences.

Each repetition is followed by short segments of "protospacer DNA" from previous exposures to foreign genetic elements. CRISPR spacers ize and cut the exogenous genetic elements using RNA interference.

The CRISPR immune response occurs through two steps: CRISPR-RNA (chNA) biogenesis and chNA—guided erence. CrRNA molecules are composed of a variable sequence transcribed from the protospacer DNA and a CRISP repeat. Each chNA molecule then hybridizes with a second RNA, known as the activating CRISPR RNA (trachNA) and together these two eventually form a complex with the nuclease Cas9. The protospacer DNA encoded n of the chNA directs Cas9 to cleave complementary target DNA ces, if they are adjacent to short sequences known as protospacer adjacent motifs (PAMs). This natural system has been engineered and exploited to introduce DSB breaks in specific sites in c DNA, amongst many other applications. In particular, the CRIPSR type II system from Streptococcus pyogenes may be used. At its simplest, the CRISPR/Ca59 system comprises two components that are delivered to the cell to provide genome editing: the Ca59 nuclease itself and a small guide RNA (gRNA). The gRNA is a fusion of a ised, site- specific chNA (directed to the target ce) and a standardised trachNA.

Once a DSB has been made, a donor template with homology to the targeted locus is supplied; the DSB may be repaired by the gy-directed repair (HDR) pathway ng for precise insertions to be made.

Derivatives of this system are also possible. Mutant forms of Cas9 are available, such as CasQDlOA, with only nickase activity. This means it cleaves only one DNA strand, and does not activate NHEJ. Instead, when provided with a homologous repair template, DNA repairs are conducted via the high-fidelity HDR pathway only. CasQDlOA (Cong L., et al. (2013) Science, 339, 3) may be used in paired Ca59 complexes designed to generate adjacent DNA nicks in conjunction with two ngNAs complementary to the adjacent area on opposite strands of the target site, which may be particularly advantageous.

The elements for making the double-strand DNA break may be uced in one or more s such as plasmids for expression in the cell.

Thus, any method of making specific, targeted double strand breaks in the genome in order to effect the ion of a gene/inducible cassette may be used in the method of the invention. It may be red that the method for inserting the gene/inducible cassette utilises any one or more of ZFNs, TALENs and/or CRISPR/Cas9 systems or any derivative thereof.

Once the DSB has been made by any appropriate means, the gene/inducible cassette for insertion may be supplied in any suitable fashion as described below. The gene/inducible cassette and associated genetic material form the donor DNA for repair of the DNA at the DSB and are inserted using standard cellular repair machinery/pathways. How the break is initiated will alter which pathway is used to repair the damage, as noted above.

The transcriptional tor protein and the inducible cassette may be supplied for the method of the invention on separate vectors. A r” is a c acid molecule, such as a DNA molecule, which is used as a e to artificially carry genetic material into a cell. The vector is generally a nucleic acid sequence that consists of an insert (such as an inducible cassette or gene for a transcriptional regulator protein) and a larger sequence that serves as the "backbone" of the vector. The vector may be in any suitable format, including plasmids, minicircle, or linear DNA. The vector comprises at least the gene for the transcriptional regulator or inducible cassette operably linked to an inducible promoter, together with the minimum sequences to enable insertion of the genes into the nt GSH. Optionally, the vectors also possess an origin of replication (ori) which permits amplification of the , for e in bacteria. Additionally, or alternatively, the vector includes selectable markers such as antibiotic resistance genes, genes for coloured markers and suicide genes.

Examples of the vectors used in the es are depicted in Figures 20 to 33.

The cell used in the method of the invention may be any human or animal cell. It is preferably a mammalian cell, such as a cell from a rodent, such as mice and rats; marsupial such as kangaroos and koalas; non-human primate such as a bonobo, chimpanzee, lemurs, s and apes; camelids such as camels and llamas; livestock animals such as horses, pigs, cattle, buffalo, bison, goats, sheep, deer, reindeer, donkeys, bantengs, yaks, chickens, ducks and turkeys; ic animals such as cats, dogs, rabbits and guinea pigs. The cell is preferably a human cell. In certain aspects, the cell is preferably one from a livestock animal.

The type of cell used in the method of the invention will depend upon the application of the cell once insertion of the genetic material into the GSH sites is complete.

Where the aim is to produce mature cell types from progenitor cells, the cell which is modified is a stem cell, preferably a pluripotent stem cell. Pluripotent stem cells have the potential to differentiate into almost any cell in the body. There are several sources of pluripotent stem cells. Embryonic stem cells (ES cells) are otent stem cells derived from the inner cell mass of a blastocyst, an early—stage preimplantation embryo.

Induced pluripotent stem cells (iPSCs) are adult cells that have been cally reprogrammed to an embryonic stem cell—like state by being forced to express genes and factors important for maintaining the defining properties of embryonic stem cells. In 2006 it was shown that the uction of four specific genes encoding transcription factors could convert adult cells into pluripotent stem cells (Takahashi, K; Yamanaka, S (2006), Cell 126 (4): 663—76), but subsequent work has d/altered the number of genes that are required. Oct—3/4 and certain members of the Sox gene family have been identified as potentially crucial transcriptional tors involved in the induction process. onal genes ing certain members of the Klf family, the Myc family, Nanog, and L|N28, may increase the induction efficiency. Examples of the genes which may be ned in the reprogramming s include Oct3/4, Sox2, Soxl, Sox3, 50x15, 50x17, Klf4, Klf2, c-Myc, N—Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, -2, Tcll, beta-catenin, Lin28b, Salll, Sall4, Esrrb, Nr5a2, Tbx3 and Glisl, and these reprogramming factors may be used singly, or in combination of two or more kinds thereof.

Where the aim is to produce stem cells with a gene knockdown or knock out for further research, such as developmental or gene function studies, the cell which is modified may be a stem cell, ably a pluripotent stem cell, or a mature cell type. Sources of pluripotent stem cells are discussed above.

If the cells modified by insertion of an inducible cassette are to be used in a human patient, it may be preferred that the cell is an iPSC derived from that individual. Such use of gous cells would remove the need for matching cells to a recipient. Alternatively, commercially available iPSC may be used, such as those available from WiCell® (WiCell ch Institute, Inc, Wisconsin, US). Alternatively, the cells may be a tissue- specific stem cell which may also be autologous or donated. Suitable cells include epiblast stem cells, induced neural stem cells and other tissue—specific stem cells.

In certain embodiments, it may be preferred that the cell used is an embryonic stem cell or stem cell line. us embryonic stem cell lines are now available, for example, WA01 (H1) and WA09 (H9) can be obtained from WiCell, and KhES-l, KhES-2, and KhES-3 can be obtained from Institute for Frontier Medical es, Kyoto University (Kyoto, Japan).

It may be preferred that the embryonic stem cell is derived without destruction of the embryo, particularly where the cells are human, since such techniques are readily available (Chung, Young et al., Cell Stem Cell, Volume 2 Issue 2 113 - 117.) Stem cell lines which have been derived without destroying an embryo are also , , available. In one aspect, the ion does not extend to any methods which involve the destruction of human embryos.

A red aspect of the present ion is the d programming of pluripotent stem cells into mature cell types. Thus, the method of the invention can be used for the manufacture of mature cell types from pluripotent stem cells. In this aspect of the invention, the inducible te for insertion into the second GSH is preferably one or more master regulators as discussed previously. These inducible cassettes may enable the cell to be programmed into a particular lineage, and different inducible cassettes will be used in order to direct differentiation into mature cell types. Any type of mature cell is contemplated, including but not limited to nerve cells, myocytes, osteocytes, chondrocytes, epithelial cells, secretory cells, and/or blood cells.

The inventors of the present application have developed a rapid, ent and scalable method for the generation of virtually any mature cell type. Such a simple and cheap method will have particular value for regenerative medicine. Previous forward programming ques utilised the Tet-On , but attempted to include all the material into one vector/site (The -one Tet-On) or tried to insert the inducible cassette into one AAVSl allele and the control system into the other AAVSl allele (DeKelver et al, 2010, Genome Res., , 1133-43 and Qian et al, 2014, Stem Cells, 32, 1230-8). Surprisingly, the dual GSH targeting method developed and described here has many unforeseen advantages. There is no potential promoter interference between the gene inserted in the first GSH and the genetic sequence of the ble cassette inserted in the second GSH. Secondly, it allows the insertion of larger cargos from the vectors, since less material needs to be inserted at each site. Thirdly, the method maximises the number of safely ed copies. Fourthly, it enables greater design flexibility. Finally, it allows for additional genetic material to be inserted, including reporter genes and miRNA switches. The method of the invention has been demonstrated to be a robust and efficient way of manufacturing mature cells from pluripotent cells.

Once the gene has been inserted into the first GSH and the ble te comprising a transgene has been inserted into the second GSH, the pluripotent stem cells may be cultured to enable forward programming to take place. These ing conditions may be specific for the type of pluripotent stem cell being used, or may depend upon the ultimate mature cell type. Whatever culturing conditions are used, the exogenous substance will control expression of the genetic sequence within the ble cassette; and may either be supplied continuously and then withdrawn in order to induce transcription or supplied as transcription is required, dependent upon its mode of action, as previously discussed.

If the aim is to program a stem cell, it may be ageous to provide that cell with extracellular prompts to aid differentiation in conjunction with the supply of inducible cassettes encoding master regulators. Cellular reprogramming strategies can be enhanced by combining master regulator or transcription factor overexpression with extracellular ling cues. Thus, it may be possible to perform systematic screen for pro-differentiation factors by modulating major signalling cascades that are implicated in development of that particular mature cell type. An instance of this is seen in Example 3.

In one aspect, the present invention provides a method for the production of es from pluripotent stem cells, comprising the steps of: a) targeted insertion of a gene encoding a transcriptional regulator protein into a first genetic safe harbour site; and b) targeted insertion of the MYODl gene operably linked to an ble promoter into a second genetic safe harbour site, wherein said inducible promoter is regulated by the transcriptional regulator protein; wherein said first and second genetic safe harbour sites are different, and culturing said cells in the presence of retinoic acid.

The MYODl gene is the gene encoding the Myogenic Differentiation 1 protein. Preferably, the retinoic acid (RA) is ans RA.

In r aspect, the present invention provides a method for the production of myocytes from pluripotent stem cells expressing MYODl, comprising culturing said cells in the presence of retinoic acid.

Preferably, the RA is all—trans RA. Preferably, the cell is overexpressing MYODl.

In a further aspect, the present invention provides a method for the production of oligodendrocytes myocytes from pluripotent stem cells, comprising the steps of: a) targeted insertion of a gene encoding a transcriptional regulator protein into a first genetic safe harbour site; and b) targeted insertion of the SOX 10 gene operably linked to an inducible promoter into a second genetic safe harbour site, wherein said inducible promoter is regulated by the riptional regulator protein; wherein said first and second genetic safe harbour sites are different, and ing said cells in the presence of ic acid.

The cells used for this may be animal or human cells. If the cells are animal, it is preferred that the animal is a livestock animal as usly defined.

The SOX-10 gene s the transcription factor SOX-10. Preferably, the retinoic acid (RA) is ans RA.

Where the cell used in the methods of the ion is pluripotent, the resultant cell may be a lineage restricted-specific stem cell, progenitor cell or a mature cell type with the d properties, by expression of a master regulator. These lineage-specific stem cells, progenitor or mature cells may be used in any suitable fashion. For example, the mature cells may be used directly for transplantation into a human or animal body, as appropriate for the cell type. Alternatively, the cells may form a test material for research, ing the effects of drugs on gene expression and the interaction of drugs with a particular gene. The cells for research can involve the use of an inducible cassette with a genetic sequence of unknown function, in order to study the controllable expression of that genetic sequence. Additionally, it may enable the cells to be used to produce large quantities of desirable materials, such as growth factors or cytokines.

In a different aspect, the cells may be used in tissue engineering. Tissue engineering requires the tion of tissue which could be used to replace tissues or even whole organs of a human or animal. s of tissue engineering are known to those skilled in the art, but include the use of a scaffold (an extracellular matrix) upon which the cells are applied in order to generate tissues/organs. These s can be used to generate an ”artificia I” windpipe, bladder, liver, pancreas, stomach, intestines, blood vessels, heart , bone, bone marrow, mucosal tissue, , muscle, skin, s or any other tissue or organ. Methods of generating tissues may e additive manufacturing, otherwise known as three-dimensional (3D) printing, which can involve directly printing cells to make s. The present invention thus provides a method for generating tissues using the cells produced as described in any aspect of the invention.

Tissues generated using cells made according to the methods of the present invention may be used for transplantation into the human or animal body. Alternatively, if the cells are from an animal, the tissues may be used for in vitro/cultured meat. The primary cell type for cultured meat is myocytes. Such tissue may, however, involve the use of a combination of cell types made according to the methods of the invention.

These may be myocytes (muscle cells), blood vessel cells, blood cells and adipocytes (fat cells). If the aim of the engineered tissue is for cultured meat, then the cell may be taken from a livestock animal.

The methods of the invention may also be performed on cells which are not pluripotent stem cells, for a y of reasons, including research, gene therapy including genetic vaccines, production of in vitro disease models and production of non-human in vivo models.

The cells used in the method of the invention may thus be any type of adult stem cells; these are unspecialised cells that can develop into many, but not all, types of cells. Adult stem cells are undifferentiated cells found throughout the body that divide to replenish dying cells and rate d tissues. Also known as somatic stem cells, they are not pluripotent. Adult stem cells have been identified in many organs and tissues, including brain, bone marrow, peripheral blood, blood vessels, skeletal muscle, skin, teeth, heart, gut, liver, ovarian epithelium, and testis. In order to label a cell a somatic stem cell, the skilled person must demonstrate that a single adult stem cell can generate a line of genetically identical cells that then gives rise to all the appropriate differentiated cell types of the tissue. To confirm experimentally that a putative adult stem cell is indeed a stem cell, the cell must either give rise to these genetically cal cells in culture, or a purified population of these cells must repopulate tissue after lantation into an . Suitable cell types e, but are not limited to neural, mesenchymal and endodermal stem and precursor cells.

Alternatively, the cells used may be a mature cell type. Such cells are differentiated and lised and are not able to develop into a different cell type. Mature cell types include, but are not limited to nerve cells, myocytes, osteocytes, chondrocytes, epithelial cells, secretory cells, and/or blood cells. Mature cell types could be any cell from the human or animal body.

Somatic stem cells and mature cell types may be ed according to the present invention and then used for applications such as gene therapy or genetic vaccination. Gene therapy may be defined as the intentional insertion of foreign DNA into the nucleus of a cell with therapeutic intent. Such a definition includes the provision of a gene or genes to a cell to e a wild type version of a faulty gene, the addition of genes for RNA molecules that interfere with target gene expression (which may be defective), provision of suicide genes (such as the enzymes herpes simplex virus thymidine kinase (HSV-tk) and cytosine deaminase (CD) which convert the harmless prodrug lovir (GCV) into a cytotoxic drug), DNA vaccines for immunisation or cancer therapy (including cellular adoptive immunotherapy) and any other provision of genes to a cell for therapeutic purposes.

Typically, the method of the invention may be used for insertion of a desired genetic sequence for transcription in a cell, ably expression, particularly in DNA es. DNA es typically encode a modified form of an infectious organism's DNA. DNA vaccines are administered to a subject where they then express the selected protein of the infectious organism, ting an immune response t that protein which is typically protective. DNA vaccines may also encode a tumour n in a cancer immunotherapy approach.

A DNA e may comprise a c acid sequence encoding an antigen for the treatment or prevention of a number of conditions including but not d to , allergies, toxicity and infection by a pathogen such as, but not limited to, fungi, viruses including Human Papilloma Viruses (HPV), HIV, HSV2/HSV1, influenza virus (types A, B and C), Polio virus, RSV virus, Rhinoviruses, Rotaviruses, Hepatitis A virus, Measles virus, Parainfluenza virus, Mumps virus, Varicella-Zoster virus, Cytomegalovirus, Epstein- Barr virus, Adenoviruses, a virus, Human T-cell Lymphoma type | virus (HTLV-I), Hepatitis B virus (HBV), Hepatitis C virus (HCV), Hepatitis D virus, Pox virus, Zika virus, Marburg and Ebola; bacteria including Meningococcus, Haemophilus influenza (type b); and parasitic pathogens. DNA vaccines may comprise a nucleic acid sequence encoding an antigen from any suitable pathogen. The antigen may be from a pathogen responsible for a human or veterinary e and in particular may be from a viral pathogen.

DNA vaccines inserted into the GSH may also comprise a nucleic acid ce encoding tumour antigens.

Examples of tumour associated antigens include, but are not limited to, cancer— ns such as members of the MAGE family (MAGE 1, 2, 3 etc.), NY-ESO-I and SSX-2, differentiation antigens such as tyrosinase, gplOO, PSA, Her-2 and CEA, mutated self—antigens and viral tumour antigens such as E6 and/or E7 from nic HPV types. Further examples of particular tumour antigens include MART—I , Melan—A, p97, beta-HCG, GaINAc, MAGE-I, MAGE-2, MAGE-4, MAGE-12, MUCI, MUCZ, MUC3, MUC4, MUC18, CEA, DDC, PIA, EpCam, melanoma antigen gp75, err 8, high molecular weight melanoma antigen, Kl 9, Tyrl, Tyr2, members of the pMel 17 gene family, c-Met, PSM (prostate mucin antigen), PSMA ate specific membrane antigen), prostate secretary protein, alpha—fetoprotein, CA 125, CA 19.9, , BRCA—l and BRCA-Z antigen.

The inserted genetic sequence may e other types of therapeutic DNA molecules. For example, such DNA molecules can be used to express a functional gene where a subject has a genetic disorder caused by a dysfunctional version of that gene. Examples of such diseases include Duchenne muscular dystrophy, cystic fibrosis, Gaucher's Disease, and adenosine deaminase (ADA) ency. Other es where gene therapy may be useful include inflammatory diseases, autoimmune, chronic and ious diseases, including such disorders as AIDS, cancer, neurological es, vascular disease, holestemia, various blood disorders including various anaemias, thalassemia and haemophilia, and ema. For the treatment of solid tumours, genes encoding toxic peptides (i.e., chemotherapeutic agents such as ricin, diphtheria toxin and cobra venom factor), tumour suppressor genes such as p53, genes coding for mRNA sequences which are antisense to transforming oncogenes, antineoplastic peptides such as tumour necrosis factor (TNF) and other cytokines, or transdominant negative mutants of transforming oncogenes, may be expressed.

Other types of eutic DNA molecules are also contemplated. For example, DNA molecules which are ribed into an active, non-coding RNA form, for example a small interfering RNA (siRNA) may be inserted.

The methods of the invention thus extend to s of knocking down nous gene expression or knocking out endogenous genes using non-coding RNAs within the inducible cassette.

Thus, the method of the invention may be used to specifically and stably insert a genetic sequence within the inducible cassette which may be controllably transcribed. This has numerous advantages in somatic stem cells and mature cell types. It allows for more closely regulated gene therapy approaches, ensuring that critical genes are not disrupted and allowing the expression of the inducible te to be turned off if any e s occur. it also allows for closely regulated endogenous gene knockdown or knockout, in order to interrogate gene function and development.

The invention extends to the cells produced by the method of the invention. The cells may be defined as being modified at a first genomic safe harbour site to include a transcriptional regulator n and at a second genetic safe harbour site to include a genetic sequence operably linked to an ble promoter which is regulated by the transcriptional regulator protein. The two GSH are different and distinct. Preferably the cells are homozygous at both insertion sites. All elements are as previously described.

The cells produced according to any of the methods of the invention have applications in diagnostic and eutic methods. The cells may be used in vitro to study cellular development, provide test systems for new drugs, enable screening methods to be developed, scrutinise therapeutic regimens, provide diagnostic tests and the like. These uses form part of the present invention. Alternatively, the cells may be transplanted into a human or animal patient for diagnostic or therapeutic purposes. The use of the cells in therapy is also included in the present invention. The cells may be allogeneic (i.e. mature cells removed, modified and returned to the same individual) or from a donor (including a stem cell line).

All documents referred to herein are hereby incorporated by nce.

Sequences AAVSl - NCBl GenBank $513291 SEQ ID No 1 :Tet02 19n sequence SEQ ID No 2 : hROSA insertion site genomic sequence SEQ ID No 3 :STDtetR-nls (nucleotide) and SEQ ID No 4 - STDtetR-nls (amino acid) SEQ ID No 5 :OPTtetR—nls (nucleotide) and SEQ ID N06 - OPTtetR-nls (amino acid) SEQ ID No 7 to 80: s from table 3.

SEQ ID No 81: Figure 18B AAVSl FWD; SEQ ID No 82: Fig 18B AAVSl REV SEQ ID No 83: Figure 188 tracer FWD; SEQ ID No 84: Fig 188 tracer REV SEQ ID No 85: Figure 19E HI POL3 FWD; SEQ ID No 82: Fig 19E HI POL3 REV This is the genomic sequence of the 6 insertion site; it includes the 5’ homology arm, the cut site (bold), and the 3’ homology arm: (SEQ ID NO 2) GCTCGAAACCGGACGGAGCCATI’GCTCTCGCAGAGGGAGGAGCGC'ITCCGGCTAGCCTCTI’GTCGCCGATTGGCCGITTC TCCTCCCGCCGTGTGTGAAAACACAAATGGCGTATTCTG G'I'I'GGAGTAAAGCTCCTGTCAGTI'ACGCCGTCGGGAGTACG CAGCCGC'I'I'AGCGACTCTCGCG'I'I'GCCCCCTGGGTGGGGCGGGTAGGTAGGTGGGGTGTAGAGATGCTGGGTGTGCGG GCGCGGCCGGCCTCCTGCGGCGGGAGGGGAGGGTCAGTGAAATCGGCTCTGGCGCGGGCGTCCTCCCACCCTCCCCTTC C'I'I'CGGGGGAGTCG ACCCGCCGCCTGCTTGTC'ITCGACACCTGA‘I‘I’GGCTGTCGAAGCTGTGGGACCGGGCCCTTG CTACTGGCTCGAGTCTCACATGAGCGAAACCACTGCGCGGGGCGCGGGGGTGGCGGGGAGGCGGGCG‘I'I'GGTACGGTC CTCCCCGAGGCCGAGCGCCGCAGTGTCTGGCCCCGCGCCCCTGCGCAACGTGGCAGGAAGCGCGCGCTGGAGGCGGGG GCGGGCTGCCGGCCGAGACTTCTGGATGGCGGCGGCCGCGGCTCCGCCCCGGG'I'I'CCCACCGCCTGAAGGGCGAGACA AGCCCGACCTGCTACAGGCACTCGTGGGGGTGGGGGAGGAGCGGGGGTCGGTCCGGCTGGTITGTGGGTGGGAGGCG C'I'I'G'I'I'CTCCAAAAACCGGCG CGAGCTGCAATCCTGAGGGAGCTGCGGTGGAGGAGGTGGAGAGAAGGCCG CACCC'I'I'C GGGGGAGGGGAGTGCCGCAATACCTITATGGGAGTI'CTCTGCTGCCTCCCGTCTTGTAAGGACCGCCCTGGGC CTGGAAGAAGCCCTCCCTCC‘I‘I’TCCTCCTCGCGTGATCTCGTCATCGCCTCCATGTCGAGTCGCTI'CTCGATTATGGGCGG GATTC'ITI'I'GCCTAGGCTI'AAGGGGCTAACTI'GGTCCCTGGGCGTTGCCCTGCAGGGGAGTGAGCAGCTGTAAGA'ITTGA GGGGCGACTCCGATTAGTTTATCTFCCCACGGACTAGAGTTGGTGTCGAGGTTATI'GTAATAAGGGTGGGGTAGGGAAA TGGAGC'I'I'AGTCA'I'I'CACCTGGGGCTGA‘I‘ITI'ATGCAACGAGACTGCGGA1TATCACTAC'I'I'ATCATTITI'GGAG CATI‘ITI' CTAGAGACAGACATAAAGCATGATCACCTGAG'I'I'I'TATACCA'ITTGAGACCCTTGCTGCACCACCAAAGTGTAGCATCAGG 'I'I'AAATCTTAATAGAAAAA'I'ITI'AGC'ITI'I'GCTI'GAGAAACCAGTGC'I'I'CCCTCCCTCACCCTCTCTCCCCAGGCTCTCTACC CCT'I'I'G CATCCCTACCAGGCATCTTAGCAACTCTCACTCATAC'I'I'GATCCCAT'I'I'I'CCATI'TG'I'I'GTAC'I'I'GCTCCTCTAGTAT TCAGACATAGCACTAGC‘I'I'I'CTCCCTCTC'I'I'GATC‘I'I'GGGTAG CCTG GTGTCTCGCGAAACCAGACAGATTGGTTCCACCAC AAATTAAGGCTI'GAGCTGGGGCTTGACTCITACCCAGCAGTGCTF'I'I'ATTCCTCCCTAG'I'I'CACGTTC'I'I'AAATG'I'I'TATC'I'I' I'CA'I'I'I'I'ATCCT'I'I'I'I'CC'I'I'AG CTGG GA'I'I'CTGTCCCTGACCGTC‘I‘I’CACAGTCCAGGTGATC'I'I'GACTACTGCTI'TA CAGAGAATTGGATCTGAGGTTAGGCAACATCTCCCTI'ITTCTI'CCTCTAAATACCTCTCAT'I'I'CTGITCTI'ACCAGTTAGTAA CTGATCTCAGATGCCTGTGTGATAGCTTCC STDtetR-nls: (SEQ ID No 3 and 4) 4o Nucleotide and amino acid sequences of the tetracycline-sensitive repressor protein (tetR) containing an N— terminal SV40 r localization signal (nls, highlighted in grey). Sequences are reported either before or after codon optimization (STDtetR and OPTtetR, respectively). Dots indicate the synonymous mutations introduced in the OPTtetR. 33173 ”33"“3‘3‘13$3,“333$3":T333333$$§§§3§€§N§Y$S§RC3§§CSXC$MRfifix‘3&3 fifi‘xxxk’VsfiRLBKSX’fi'KRSfiigg-fi ‘3‘&§3M§b3"{3333333°3N£>$SA‘3‘??R§3§§R€f"$333333?{33:52:32-R§§3§S€§$§i§§‘i§§3fi$§§€k iaREVGXEGEEfiE‘RRfiRQXEGVfiSQ 3333333333333333333333333333:33:3:3:.33:*3“:333 3:33.3:::33: §¥L¥Kfi¥fifififififi£fi£fii$fih &&?$51‘$”fi§\§33:?{1fiki'3‘31‘3‘3‘312x’ffi’3‘3RfikkfiGMMKKfi‘i’€3fif3§3€§3§3133’-3‘3“‘3K£w3RAT§RQ$ 33 3333v333333333 33333333333333333333333“3333333323333:3333333333333333333333:33333.3333: 3:3: ”€83?RCR£L$§£R§G3X'3’SLG§RF 3333333333333:333‘3333““3:333:3W‘333‘91‘33‘3‘3‘33w3‘33333$333333“?33:33:33,333 3333333333333333333333 3333333333333333333333333333;332:3333-3333:333:“”3333:3383:???3”“:€333€333 ‘3333333333333333332333333 3333: 33333333:33333333333333.3333333333323333332333“: 3"!“3‘333323‘3‘2333::333333::33:3 33.33333313333333333333333 3333::33-3:3333“3“\\*“333:*233.3333“33:3:33333:3:¢33333:3W331‘33333:3333 *::*3:33***333333m3 33333333333333.3333333323, ‘ ’3‘; ‘32::333333333333“333333333333333333333333333333333)”“‘3 333333333333333 The sequence for optimised tetR: OPTtetR-nls (SEQ ID NO 5 and 6): The invention will now be described in relation to the following non-limiting examples: Examples Materials and Methods used in the es: hPSC maintenance culture and germ layer differentiation Feeder— and serum-free hESC (H9 line; WiCell) and hiPSC (Cheung et al, Nat. Biotechnol. 30, 165—173 (2012)) culture was performed. Briefly, cells were plated on n/MEF media-coated culture dishes [MEF-media consisted of Advanced DMEM/F12 (90%, Gibco), fetal bovine serum (10%, Gibco), L-Glutamine (1 mM, Gibco), 2-Mercaptoethanol (0.1 mM, Sigma-Aldrich) and Penicillin/Streptomycin (1%, Gibco)], and cultured in chemically defined media [CDM, consisting of IMDM (50%, Gibco), F12 (50%, Gibco), concentrated lipids (100x, Gibco), monothioglycerol (450 uM, Sigma-Aldrich), n (7 ug/ml, Roche), transferrin (15 ug/ml, Roche), bovine serum albumin fraction V (5 mg/ml), and Penicillin/Streptomycin (1%)] supplemented with 10ng/ml Activin-A and 12ng/ml F6 F2. Cells were passaged in small clumps using collagenase every 5-6 days.

Differentiation of hPSCs into the germ layers was induced in adherent hESC cultures according to previously published ed differentiation protocols for endoderm, lateral plate mesoderm, and neuroectoderm (Touboul, T. et al. Hepatology 51, 1754—1765 (2010), Cheung et al, (2012) and Douvaras, P. et al. Stem Cell Reports 3, 250—259 (2014).) Briefly, definitive endoderm was derived by culturing hPSCs for 3 days in CDM- PVA (without insulin) supplemented with FGF2 (20ng/ml), Activin-A (100ng/ml), BMP4(10ng/ml, Marko Hyvonen, Dept. of Biochemistry, University of Cambridge), and LY-294002 (10 uM, Promega) 3. For derivation of neuroectoderm, hPSCs were cultured for 6 days in CDM—BSA mented with SB—431542 (10 uM, Tocris), LDN-193189 (0.1 uM, Tocris) and RA (0.1 uM, Sigma) 4. Lateral plate mesoderm was obtained by culturing hPSCs for 36h in CDMPVA supplemented with FGF2 (20 ng/ml), l BMP4 (R&D), and LY294002 (10uM), and for 3.5 subsequent days in CDM-PVA supplemented with FGF2 ml) and BMP4 (50ng/ml).

Differentiation of hESCs. Differentiation was initiated in adherent cultures of hESCs 48h following ing.

Media changes were generally performed daily, and volumes were adjusted for cell density. Mature cell types were obtained using methods previously bed in the art. Mature cell types obtained included neural cells, osteocytes, ocytes, smooth muscle, c fibroblasts, cardiomyocytes, intestine, pancreas, cytes, giocytes or lung.

Gene targeting constructs and molecular cloning Design and construction of the 6 gRNA and Ca59n expression plasmids is bed here: A CRISPR/Ca59n based strategy to specifically target the hROSA26 locus and to insert inducible cassettes using homologous recombination. To induce a genomic DSB at the correct integration site, a CRISPR/Cas9 nickase system was designed. In contrast to the commonly used wild-type Ca59 se which is let by a single gRNA to its genomic target site, the D10A mutant Ca59 nickase (Ca59n) is directed by a pair of riately designed gRNAs to simultaneously uce single—stranded cuts on both strands of the target DNA. This strategy effectively doubles the number of bases required for genome editing and thereby increases icity. The web—based software ”CRISPR Design Tool” was used to define potential target sites for chNA—guided nucleases that are close to the integration site. Within a ce stretch of 250 bp around the target site (125 bp on each site of the actual integration site), the top hit yielded a pair of gRNAs that collectively reached a ”high quality” score of 97, with no predicted off target effects. The gRNAs [gRNA—A 5’- GTCGAGTCGCTTCTCGATTA-(TGG)-3’ and gRNA—B 5’-GGCGATGACGAGATCACGCG-(AGG)-3’ (PAM sites in parenthesis) were synthesised de novo and ligated into expression s. The final plasmids encode for either of the two gRNAs, respectively, and the Ca59n D10A—mutant (Figures 20 and 21).

A donor plasmid was constructed that serves as a template DNA to facilitate homology ed repair of a Cas9n-induced DSB. Two hROSA26 homology arms were generated by high-fidelity PCR amplification.

Genomic DNA that was ed from H9 hESCs served as a template. The 5’ and 3’homolgy arms were 904bp and 869bp in length, respectively. Both were subsequently inserted into the multiple cloning site of the pUC19 vector. To target the 6 locus, cells were transfected with the plasmid, the two gRNA/Ca59n construct and the EGFP donor plasmid (Figure 22) The AG-rtTA targeting vector (figure 23)was constructed by cloning the coding sequence of a third tion rtTA (PCR-amplified from pLVX—Tet3G) into the BamHI/Mlul sites of AG-EGFP thus replacing the EGFP sequence. AAVSl ZFN expression plasmids were a generous gift of Dr. Kosuke Yusa ome-Trust Sanger Institute). The ble EGFP AAVSl targeting vector was constructed by Gibson Assembly (New d Biolabs) in which three inserts were ligated into the EcoRI/Hindlll sites of the multiple cloning site of the pUC19 vector (Thermo Fisher Scientific): The first insert comprised the upstream AAVSl homology arm, a splice acceptor, a te and the puromycin resistance cassette (PCR-amplified from pTRE-EGFP; addgene 22074, deposited by Jaenisch). The second insert contained the inducible TRE3G promoter (PCR- amplified from pLVX—TREBG). The third insert comprised the EGFP expression cassette and the AAVSl downstream homology arm (PCR—amplified from pTRE-EGFP; addgene 22074, deposited by RudolfJaenisch).

The resulting plasmid was termed pAAV_TRE-EGFP (Figure 32). The pAAV_TRE—NGNZ and pAAV_TRE- MYODl(Figure 33) targeting vectors were constructed by cloning the NGN2 and MYODl coding sequence, respectively (NGN2: PCR-amplified from RE—NGN2, gift from Oliver BrUstle; MYODl: PCR—amplified from a commercially available cDNA plasmid, Open Biosystems MH56278-202832821, Accession: BC064493, Clone ID: 5022419) into the Spel/EcoRl sites of pAAV_TRE-EGFP, thus replacing the EGFP sequence.

Further plasmids were also created using similar s, and all ds used are depicted in Figures 20 to 33. These ds were either created or generously donated. The plasmids used in the es include (in order of figures 20 — 33): pSpCa59n(BB),_R26-R, pSpCa59n(BB) (the combination of these two plasmids is predicted to induce a specific double strand break in the intron between exons 1 and 2 of THUMPDS3-ASl on chromosome 3 (ROSA26 |ocus)),_R26-L pR26_CAG_EGFP, pR26_CAG_rtTA, pZFN-AAVSl-L-ELD (zinc finger nuclease left), pZFN-AAVSl-R-KKR (zinc finger nuclease right), pAAV_CAG_EGFP (donor), pR26-Neo_CAG- OPTtetR (hROSA26 targeting of codon-optimized tetR), pAAV-Puro_iKD (AAVSl targeting of inducible sh RNA), pAAV-Neo_CAG-Ca59 (AAVSl targeting of Cas9), pAAV-Puro_siKO (AAVSl targeting of inducible , pAAV- Puro_siKO-2TO (AAVSl targeting of inducible gRNA, version with 2 tet operons in promoter), pAAV_TRE-EGFP (EGFP inducible overexpression, attached) and pAAV_TRE-MYODl (MYODl inducible overexpression for muscle).

Gene targeting Targeting of the hROSA26 locus and the AAVSl for gene knockdown and knockout was performed by nucleofection. Human otent stem cells (PSCs) were iated to single cells with TrprE Select (Gibco), and 2x106 cells were nucleofected (100ul reaction ; total of 12ug of DNA, which was equally divided between the two gRNA/Ca59n plasmids and the targeting vector) using the Lonza P3 Primary Cell 4D- Nucleofector X Kit and cycle CA—137 of the Lonza 4D-Nucleofector System. Nucleofected hPSCs were plated onto irradiated multi-drug resistant (DR4) mouse embryonic fibroblasts and cultured in KSR media [consisting of Advanced DMEM/F12 (80%), knock-out serum replacer (20%, Gibco), L-Glutamine (1 mM), 2— Mercaptoethanol (0.1 mM) and Penicillin/Streptomycin (1%)] supplemented with FGF2 (4ng/ml, Department of Biochemistry, University of Cambridge). Y-27632 (5 (1M, Tocris) was added for 24h before and after nucleofection to promote cell survival. After 3-6 days, neomycin-resistant hPSCs were selected by adding G418 (50 ug/ml, Sigma-Aldrich) for 7—10 days. Subsequently, individual clones were picked, expanded in feeder—free conditions and finally ed by genotyping.

Targeting of the AAVSl locus was also performed by lipofection. Human PSCs were seeded in feeder-free conditions in 6—well plates, and transfected 48h after passaging. Transfection was med in Opti-M EM (Gibco) supplemented with Lipofectamine2000 (10 ul/well, Thermo Fisher Scientific) and a total of 4 ug of DNA (equally divided between the two AAVSl ZFN plasmids and the ing ) for 24h. After 3—5 days, resistant hPSCs were selected by adding puromycin (1 ug/ml, Sigma-Aldrich) for 5-8 days. Subsequently, individual clones were picked, expanded and ed by genotyping. Antibiotic ance can be used to select clonal lines.

Drug—resistant hPSC clones from targeting experiments were screened by genomic PCR to verify site-specific ble cassette ation, to determine the number of targeted alleles, and to exclude off-target integrations. PCRs were performed with LongAmp Taq DNA Polymerase (New England Biolabs). Table 2 reports the primer combinations used for the various targeting vectors. The results of all targeting experiments are summarized in Table 1. Karyotype analysis was performed by standard G banding techniques (Medical Genetics e, Cambridge University als). To e the targeted human PSCs for chromosome analysis, cells were incubated in fresh culture media supplemented with Y-27632 (5 uM, Tocris) and KaryoMAX Colcemid (100 ng/ml, Gibco) for 4h at +37°C. Subsequently, cells were harvested as single cells, washed, and pelleted. Nuclei swelling and spreading of the chromosomes was achieved by treatment with hypotonic 0.055 M KCl-solution for 5-10 minutes. Finally, cells were fixed with methanol and glacial acetic acid (ratio 3:1).

For , AAVSl targeting was performed by lipofection as previously described. Briefly, hPSCs were seeded feeder—free in 6—well plates, and transfected 48 h following cell passaging with 4 pg of DNA (equally divided between the two AAVSl ZFN plasmids and the targeting vector) using 10 ul per well of ctamine 2000 in Opti—M EM media (Gibco) for 24 h, all according to manufacturer’s instructions. After 4 days, 1 pg ml-1 of Puromycin was added to the culture media, and individual clones were picked and expanded following 7-10 days of selection.

For single site OPTiKO, AAVS1 ing was performed by nucleofection. hESCs pre-treated for 16 h with 10 pM Y-27632 s) were dissociated to clumps of 2-8 cells using Accutase (Gibco), and 2 x 106 cells were nucleofected in 100 pl with a total of 12 pg of DNA (4 pg each for the two ZFN plasmids, and 2 pg each for the two targeting vectors) using the Lonza P3 Primary Cell 4D-Nucleofector X Kit and the cycle CA-137 on a Lonza 4D-Nucleofector System, all according to manufacturer’s instructions. Nucleofected hESCs were plated onto a feeder layer of irradiated DR4 (puromycin and neomycin resistant) mouse embryonic fibroblasts and cultured in KSR media supplemented with 4 ng ml-1 FG F2 and 10 pM Y-27632 (this last only for the first 24 h). After 4 days, hPSC es ng both puromycin and neomycin resistance gene were selected for 7—10 days with pg ml-1 of Geneticin (G418 Sulfate, Gibco) and 0.5 pg ml-1 Puromycin. Individual clones were then picked and expanded in feeder-free conditions.

AAVSl—EGFP, ROSA26-EGFP, ROSA26-STDtetR, ROSA26-OPTtetR, and ROSA26-EGFPd2 hESCs were ted by lipofection (AAVS1 locus) or nucleofection (ROSA26 locus) of the targeting vectors with AAVS1 ZFN or ROSA26 CRISPR/CasQn pairs (as described above). 2 pg ml-l Blasticidin S—HCI (Gibco) was used for pR26- Bsd_CAG-EGFPd2 plasmid. Generation of inducible EGFP overexpression hESCs carrying ROSA26-rtTA and TRE-EGFP enes is described elsewhere. Briefly, cells were sequentially gene ed first by nucleofection of pR26-Neo_CAG-rtTA with ROSA26 CRISPR/Cas9n plasmids, then by lipofection of pAAVPuro_ TRE-EGFP with AAVSl ZFN plasmids.

Gene targeted hPSC clonal lines were screened by genomic PCR to verify site-specific targeting, determine the number of alleles targeted, and exclude off-target integrations of the targeting plasmid (see Fig. 16A).

Inducible cassette overexpression Overexpression of inducible cassettes (EGFP, NGN2, MYODl and SOXlO, respectively) was induced by adding doxycycline hyclate (Sigma—Aldrich) to the culture media. Unless stated otherwise cline was used at a final concentration of 1 pg/ml. Media ning doxycycline was kept light protected, and changed every 24 hours. Cells expressing EGFP are herein termed OPTi-EGFP, those expressing NGN2 are termed OPTi-NG N2, cells sing MYODl are called OPTi-MYODl and cells expressing OLIG2-SOX10 are called OPTi- OLIGZ- SOX10.

Inducible gene knockout and knockdown Unless otherwise described in the figure legends or Examples, tetracycline hydrochloride (sigma—Aldrich) was used at 1 pg ml'1 to induce gene knockdown or knockout. Induction of neurons Pluripotent OPTi—NGNZ cells were dissociated into single cells with TrprE and plated onto Matrigel (35 pg/cmz, Scientific Laboratory es) coated dishes at a density of 75.000 cells per well of a 12 well plate.

Forward programming was initiated 24-48 hours after the split. Unless stated otherwise, the induction was performed in DMEM/F12 (Gibco) supplemented with Glutamax (100x, Gibco), Non-Essential Amino Acids (100x, Gibco), 2—Mercaptoethanol (50 pM), llin/Streptomycin (1%), and doxycycline (1 pg/ml). After 2 days of induction, the medium was switched to Neurobasal—medium supplemented with Glutamax (100x), 827 (50x, Gibco), BDNF (10 ng/ml, Peprotech), NT3 (10 ng/ml, R&D Systems), Penicillin/Streptomycin (1%), and doxycycline (1 ug/ml).

Induction of skeletal myocytes Pluripotent OPTi-MYODl cells were dissociated into single cells with TrprE and plated onto gelatine/MEF- medium coated dishes at a density of 100.000 cells per well of a 12 well plate. Forward programming was initiated 24-48 hours after the split. Unless stated otherwise, the induction was performed in DMEM (Sigma- Aldrich) supplemented with L-Glutamine (2 mM), Z—Mercaptoethanol (50 uM), llin/Streptomycin (1%), insulin (7 ug/ml), all-trans retinoic acid (1 uM, Sigma-Aldrich), and doxycycline (1 ug/ml). After 5 days of induction, the medium was supplemented with 021 (3 uM, Tocris) and heat-inactivated horse serum (2%, Gibco) to enhance maturation.

Induction of Oiigodendrocytes Pluripotent OLIGZ-ZA—SOXlO OPTi-OX hPSCs were grown in es on gelatine/MEF coated culture dishes.

Before the start of induction they were treated with SB and LDN overnight. The following day induction was initiated in CDM supplemented with cline (1 ug/ml) and RA (0.1 uM). One day after induction, cells were split in CDM supplemented with RA (0.1 (1M), PM (1 (1M), and Y—27632 (5 uM), PDGFaa (20 ng/ml, Peprotech), FGF2 (5 ng/ml) onto PDL/laminin coated culture dishes (100.000 cells per well of a 12 late).

The ing day cells were switched to oligodendrocyte media consisting of DMEM/F12, mented with ax (100x), Non-Essential Amino acids (100x), 2-Mercaptoethanol (1000x), Penicillin-Streptomycin (100x), N2 Supplement (100x), BZ7 Supplement (50x), Insulin 7 ug/ml (Marko Hyvonnen), T3 60 ng/ml (Sigma), Biotin 100 ng/ml (Sigma), db-cAMP 1 uM (Sigma). Oligodendrocyte medium was supplemented with dox (1 ug/ml), PDGFaa (20 , FGFZ (5 ng/ml), RA (0.1 uM) and PM (1 (1M). Seven days post ion RA and PM was awn. To keep induced cells in a proliferative state, cells were passaged every 4 days (75.000 cells per well of a 24 well plate) in the continued presence of the mitogens PDGFaa and FGFZ. For differentiation of proliferative oligodendrocyte precursors, PDGFaa and FGFZ were withdrawn. Human recombinant NT3 (5 ng/ul, R&D Systems) was added to enhance cell survival.

Quantitative real-time PCR (qPCR) RNA was extracted using the GenElute Mammalian Total RNA Miniprep Kit and the On-Column DNAseI Digestion Set (Sigma-Aldrich). cDNA synthesis was performed with the Maxima First Strand cDNA Synthesis Kit (Thermo Fisher ific). d Biosystems SYBR Green PCR Master Mix was used for qPCR. Samples were run on the Applied Biosystems 7500 fast PCR machine. All samples were analyzed in technical ates and normalized to the house-keeping gene Porphobilinogen Deaminase 1 (PBG D). Results were analyzed with the AACt method. See Table 3 for primer sequences.

Flow cytometry For analysis of EGFP expression cells were harvested with TrprE Select (Gibco) for 5-10minutes at 37°C to obtain a single cell suspension. Following a wash with PBS, cells were ended in ice-cold PBS supplemented with DAPI (10 ug/ml), and incubated for 5 minutes on ice. Cells were analyzed using a Cyan ADP flow-cytometer to determine the levels of EGFP expression of viable cells (DAPI negative). For staining and analysis of myosin heavy chain expression cells were harvested with TrprE Select (as for EGFP expression analysis), washed once with PBS, and fixed and permeabilized with Cytofix/Cytoperm solution (BD Biosciences). Subsequently, cells were washed and blocked in Perm/Wash buffer (BD Biosciences) supplemented with 3% bovine serum albumin (BSA) at +4°C ght. Staining with a PE-conjugated anti-MYH antibody (table 4) was carried out in Perm/Wash buffer for 1h at +4°C in the dark. After three washes with Perm/Wash buffer cells were analyzed with a Cyan ADP flowcytometer to determine the levels of MHC expression. Data analysis was performed with FlowJo (v10) and Graphpad Prism (v6).

Western blot Whole-cell protein was extracted with CelLytic M (Sigma-Aldrich) supplemented with complete Protease Inhibitor (Roche), and subsequently quantified by using n Quantification Kit-Rapid (Sigma-Aldrich).

Protein electrophoresis was performed with NuPAGE LDS Sample Buffer and 4—12% NuPAGE Bis—Tris Precast Gels rogen). Following protein transfer on PVDF, membranes were blocked with PBS supplemented with 0.05% Tween—20 (PBST) 4% milk for 1h at room temperature, and incubated with y antibodies overnight in PBST 4% milk. Membranes were washed with PBST, incubated with HRP-conjugated secondary dies (Sigma-Aldrich) in PBST 4% milk, incubated with Pierce ECL2 Western ng Substrate (Thermo Fisher Scientific), and exposed to X-Ray Super RX Films (Fujifilm).

Immunocytochemistry Cells were fixed in 4% paraformaldehyde (diluted in PBS) for 20 minutes at room temperature and subsequently washed three times with PBS. The cells were then blocked with 10% donkey serum (Sigma- Aldrich) and permeabilized with 0.3% Triton X-100 (diluted in PBS) for 20 minutes at room temperature. uently, cells were incubated with riately diluted primary antibodies (supplemental experimental procedures) in 2% donkey serum and 0.1% Triton X-100 (diluted in PBS) at 4°C overnight. Triton-X was omitted throughout all steps when staining the surface antigen PDGFRA, A2B5, and 04. After three washes with PBS, the cells were incubated for 1 hour at room ature with corresponding donkey fluorophore-conjugated secondary antibodies (Alexa Fluor 488, 555, 568, and/or 647) in PBS supplemented with 1% donkey serum.

Nuclei were visualized with 4',6—diamidino-Z-phenylindole (DAPI, Thermo Fisher Scientific). EGFP expression and stainings were imaged using a Zeiss LSM 700 confocal microscope (Leica). The percentage of Bill- n positive cells was calculated by ining BIII-tubulin expression in at least 50 randomly selected DAPI—positive cells in 3 visual fields of 3 biological replicates using an inverted s |X71 fluorescence microscope. tical analysis was performed with Graph Pad Prism (v6). The number of replicates, the statistical test used, and the test results are described in the figure legends. Unless stated otherwise data is presented as mean i SEM.

Example 1: Dual targeting of EGFP To develop an inducible pression platform in hPSCs, we sequentially targeted the two components of the Tet-ON system into two different GSHs. A constitutively expressed third generation rtTA was targeted into the human ROSA26 (hROSA26) locus by using a CRISPR/CasQn-based targeting strategy and an inducible EGFP inducible cassette was inserted into the AAVSl (Fig. 1a; Figures 4a — c). Both hROSA26 and AAVSl targeting was highly efficient (Figures 4d—f, Table 1) and did not affect hPSC genomic stability, self-renewal, and differentiation (data not shown), ore arguing against rtTA-dependent cellular toxicity.

We then selected dual GSH-targeted clones that carried either one or two copies of each of the two inducible cassette (Figure 5a). Homozygous targeting of the rtTA resulted in approximately two—fold higher levels of rtTA protein (Figure 5b), and also in significantly increased EGFP levels following induction, when compared to heterozygous rtTA sion es 5c—5e). Additionally, clones with homozygous targeting of the inducible EGFP cassette showed higher and more homogeneous EGFP levels ed to lines with heterozygous targeting (Figures Sc— 5e). Importantly, all correctly targeted lines showed robust inducible EGFP expression, which was at least twenty-fold higher compared to the strong constitutive CAG promoter (Fig. 1b, Figures 5c- e). Collectively, these results t our initial hypothesis that targeting two copies of both elements of the Tet-ON system would result in maximal expression following ion. The peak of EGFP levels was reached approximately four days after induction, and expression was quickly reversed upon doxycycline awal (Fig. 1c). Moreover, EGFP expression could be titrated by adjusting the dose of doxycycline (Fig. 1d).

Importantly, inducible EGFP expression was not only highly efficient in hPSCs, but also during differentiation into the germ layers (colour photographic data not shown, data on Figures 6a-6d). Finally, and in agreement with the known tight transcriptional control of third generation Tet—ON systems, there was no detectable background sion of EGFP mRNA or protein in the absence of doxycycline as determined by flow try and qPCR, respectively (Fig. 1b, Figure 6d). Overall, these results ished that dual GSH targeting of the Tet-ON system is a powerful strategy for optimal expression of inducible cassettes in hPSCs and their derivatives.

Example 2: Derivation of tory cortical neurons from hESC and hiPSC Previous studies have shown that these cells can be readily derived by lentiviral overexpression of any of the uronal bHLH-factors (ASCLl, NGN2, or NEURODl) in hPSCs. Therefore, we generated OPTi- NGN2 hPSCs (Fig. 2a, Table 1). NGNZ induction ed in rapid downregulation of pluripotency factors (Figure 7) and tion of a neuronal transcriptional program (Fig. 2b). Induced cells exhibited neuronal processes as early as three days post ion (data not shown). After one week, all cells yed a neuronal logy and expressed pan-neuronal marker proteins, such as Blll-tubulin and MAP2 (Figure 2c). Quantitative RT-PCR revealed strong induction of typical forebrain markers such as BRNZ and FOXGl, and of glutamatergic s including GRIA4 and VGLUTZ (Fig. 2b), indicative of an excitatory cortical neuronal identity. Collectively, these results demonstrated a ic improvement in both speed and efficiency in generating neurons compared to traditional hPSC differentiation protocols, and a substantial increase in efficiency and purity relative to both transdifferentiation and lentiviral-based forward programming protocols. Similar results were obtained with OPTi—NGNZ hiPSCs, confirming the robustness of this method. Finally, we did not observe any drop in the efficiency of neuronal induction over extended e periods of Opti-NGN2 hPSCs (>25 passages, Figure 2c).

Overall, our results demonstrated that OPTi-NGNZ hPSCs can be used as an inexhaustible source for unlimited, highly scalable, rapid, single step, virus-free, and near-deterministic generation of neurons.

Example 3: Generation of al myocytes The transcription factor MYOD1 is known to induce myogenic transdifferentiation when overexpressed in a variety of somatic cell types, however, the y of hPSCs to o induced myogenic forward programming is currently debated. We generated OPTi -MYOD1 hPSCs (Table 1), but we noted that induction of MYODl expression following doxycycline treatment resulted in near complete cell death within 3-5 days in a broad range of culture ions that were suggested previously to facilitate the conversion of hPSCs into al myocytes. Since it is widely established that cellular reprogramming gies can be enhanced by combining transcription factor overexpression with extracellular signaling cues, we med a systematic screen for pro—myogenic factors by modulating major signaling cascades that are implicated in primitive streak formation, somitogenesis, and esis. We found that the addition of all—trans retinoic acid (RA) in conjunction with MYODl overexpression resulted in rapid and near-complete conversion into myogenin and myosin heavy chain (MHC) double-positive myocytes by day 5 after induction. The effect of RA was concentration dependent and mediated through the RA—receptor isoforms RARa and RARB, consistent with the expression pattern of RA receptors during developmental myogenesis (Figure 8). This effect is thought to be independent of the mechanism of MYODl pression. Induced skeletal myocytes presented a typical spindle-like, elongated morphology, underwent ive cell fusion and exhibited strong myogenic marker expression on both mRNA and protein levels (Fig. 3b, Figure 9a — 9c). Addition of nanomolar concentrations of acetylcholine (ACh) or the selective ACh—receptor agonist carbachol resulted in complete muscle fiber contraction, demonstrating functionality of the induced myocytes. Similar results were obtained with Opti— MYODl hiPSCs (data not shown). antly, myogenic ion efficiency did not decrease over extended culture periods (>50 passages, Fig. 3d), thus demonstrating the ness and reproducibility of this method.

Finally, we noted that the levels of the MYODl—inducible cassette positively correlated with conversion efficiency, which highlights the importance of a robust gene—delivery and the ority of this method over 3O lentivirus-mediated reprogramming approaches (Figure 10). Overall, the OPTi—MYODl forward mming strategy is approximately seven times faster and five times more efficient than most recent differentiation protocols of hPSCs into skeletal myocytes. Compared to previous forward programming protocols (Tanaka, A. et al. PLoS One 8, e61540 (2013) and Abujarour, R. et 0/. Stem Cells Trans]. Med. 3, 149—60 (2014)) it is more efficient (>95% vs. 30—80%), free of ly ed ble cassettes, chemically defined, fully reproducible, and more scalable.

These findings demonstrate that this method of controlling inducible cassette expression in hPSCs can be used as inexhaustible source for high-throughput and large-scale manufacturing of homogeneous cell populations.

The speed of induction and the purity of the desired target cells are currently unrivalled by other methods.

Example 4: Generation of Oligodendrocyte precursors and Oligodendrocytes: OPTi—OX hPSCs bearing inducible SOXlO either alone or in combination with OLIGZ in form of a bicistronic expression cassette. Although cells induced with SOXlO alone ly sed the Oligodendrocyte precursor (OPC) marker 04 after 10 days of induction, these cells failed to differentiate further into myelin- expressing cells and ssively died. In contrast, the SOXlO double-overexpressing cells readily progressed from an O4—positive progenitor stage into a mature CNP/MBP-positive phenotype at 20 days post induction. Moreover, additional marker protein expression analysis confirmed that OPTi-OLIGZ-SOXlO hPSCs d in Oligodendrocyte media (Douvaras et al. 2014) supplemented with the mitogens PDGFaa and FGF2 first passed through an OPC-like stage in which they were highly proliferative and in which they co-expressed PDGFRA, AZBS, and 04. These cells were highly proliferative and could be maintained for at least three passages (Fig 12b) by ing them in the presence of mitogens. We ore named these cells i-OPCs, for induced OPCs. Remarkably, following withdrawal of mitogens and in the continued presence of doxycycline, i- OPCs readily entiated in approximately one week into mature oligodendrocytes sing the major myelin proteins CNP, PLP, MAG, M06 and MBP (Fig 12c-12d) that were capable of myelin sheath formation (data not shown). tively, these results demonstrated that the invention allowed the development of a novel, robust and rapid hPSC forward programming protocol for the generation of Oligodendrocyte precursors and oligodendrocytes.

Table 1: Summary of Genotyping results: ROSA26 236/5? 2/3/1* 7/:2/3 5/3/6* 8/8*/14 1/0/3* / 9:283/ mull-“II- 24* * 46* 92* “II-[Illn- “III-“- -I------m “III-“III- (a) Incorrect targeting: No evidence of targeting (lack of bands in 5’- and 3’-integration PCR and presence of WT band in locus PCR) or evidence of targeting, but incorrect size of 5'— or 3’-integration PCR. (b) Correct on—target integration with additional random ation of the plasmid (bands in 3’-backbone PCR). (c) Correct on—target integration (HET, zygous; HOM, homozygous). (d) Percentage of clones with t on—target integration (without additional off-target integration) (e) Percentage of clones with correct on-target integration (with or without additional off-target integration) * The three numbers are from three different targeting experiments in hESCs.

Table 2: List of primers used for genotyping PCR GAGAAGAGGCTGTGCTFCGG Locus PCR ACAGTACAAGCCAGTAATGGAG GAGAAGAGGCTGTGC'I'I'CGG -INT PCR AAGACCGCGAAGAG‘ITI'GTCC hROSA26 GAAACTCGCTCAAAAGCTGGG 3-INT PCR Genome (3') ACAGTACAAGCCAGTAATGGAG GAAACTCGCTCAAAAGCTGGG 3 BB PCR Vector Backbone (3') TGACCATGA'I'I'ACGCCAAGC Genome (5') CTGTITCCCCTTCCCAGGCAGGTCC Locus PCR Genome (3') GGAACGGGGCTCAGTCTGA Genome (5') CTG'ITI'CCCCTTCCCAGGCAGGTCC ' INT PCR TCGTCGCGGGTGGCGAGGCGCACCG AAVSl inducible cassette specific sequence 3'-INT PCR Genome (3 ') TGCAGGGGAACGGGGCTCAGTCTGA Inducible cassette ble cassette ic sequence 3'-BB PCR Vector Backbone (3') ATGCTTCCGGCTCGTATGTT Table 3: List of s for quantitative PCR CCCTGGGTGTITGCCCAGAT ACCACGGGGTACG'I'I'GTACT CAACCAGATCGGGGCCAAGTI' TUBB3 CCGAGTCGCCCACGTAGTF Table 4: List of antibodies -gM Millipore MAB312 1300 ACTN2 (a- mouse IgGl monoclonal Sigma A7811 1: 200 rabbit lgG monoclonal Abcam ab32362 1:500 (desmin) mouse IgGl monoclonal DSHB FSD 1:100 (myogenin) rabbit IgG monoclonal Abcam ab124800 1:500 (myogenin) (myosin mouse lgGZb monoclonal DSHB MFZO 1:100 heavy chains) NCAM IgGl DSHB 5.1H11 1:100 TN NT2 mouse IgGZa monoclonal DSHB CT3 1:100 (troponin T) TetR (tet mouse lgGl monoclonal Clontech 631131 1:1000 (WB) repressor) TU BA4A 1:10000 mouse lgGl monoclonal Sigma T6199 (0L4—tubulin) (WB) TU BB3 (BIII- mouse lgGl monoclonal Millipore MABl637 1:1000 tubulin) e 5: TET-ON inducible knockdown system Development of an optimized ble knockdown platform in hPSCs.

We generated hESC lines in which an EGFP ene could be ed in an inducible fashion (Fig. 143). For that, we targeted: (1) a tR expression cassette into the ROSA26 locus; and (2) a CAG-EGFP transgene plus an inducible EGFP shRNA te into the AAVSl locus (Fig. 14A,B). To express higher levels of the tetR protein to more strongly repress shRNA expression in the absence of tetracycline. For this, we performed a multi-parameter RNA and codon optimization of the bacterial tetR cDNA, and used the resulting codon- optimized tetR (OPTtetR) to generate new EGFP inducible knockdown hESC lines (Fig. 143). This modification d a ten-fold increase in the tetR expression when compared to the standard sequence (STDtetR; Fig. 14D). Further, homozygous expression of the OPTtetR was sufficient to completely prevent shRNA leakiness while fully preserving efficient knockdown induction (Fig. 14C). Of note, the inducible knockdown was rapid, reversible, and dose responsive (Fig. 14E,F). Finally, inducible hESCs displayed a normal karyotype (data not shown), trating that the genome engineering necessary to create these lines did not alter their genetic stability.

Based on these encouraging results, we further validated this method in the context of nous genes by generating hESCs carrying inducible shRNAs against POU5F1/OCT4 or BZM (data not shown). Remarkably, all the sublines analysed (6 for each gene) showed robust inducible knockdown with no significant shRNA leakiness. Tetracycline titration identified optimal concentrations to partially or fully own OCT4. As expected, a strong decrease in OCT4 ically resulted in loss of otency and induction of ctoderm and definitive endoderm markers. Similar results were obtained with 20 additional OCT4 inducible knockdown hESC sublines, confirming the robustness and reproducibility of this method.

Importantly, the generation of hESCs with strong and tightly ted knockdown was so efficient that phenotypic analyses could be performed ately after antibiotic selection on a mixed population of cells, thereby entirely bypassing the need of picking individual colonies for clonal ion. Overall, these results establish that dual targeting of GSHs with an optimized inducible own system is a powerful method to control gene expression in hPSCs. This ch is hereafter named OPTiKD, for OPTimized inducible KnockDown (Fig. 14A).

Example 6 The capacity to knockdown genes in a variety of differentiated cells would represent a significant advance over previous systems for inducible gene knockdown. To thoroughly test this possibility, we analysed the efficacy of the OPTiKD platform to knockdown an EGFP transgene in hPSCs differentiated into the three germ layers, as well as in a panel of thirteen fully differentiated cell types (Fig. 15A). For both methods, qPCR analyses demonstrated strong and ble knockdown of EGFP transcripts in all lineages tested (Fig. 17). Microscopy observations confirmed robust decrease in EGFP protein expression, and flow cytometry showed a decrease of EGFP fluorescence by more than 70% for most lineages (data not .

Example 7 Development of an optimized inducible CRISPR/Ca59 knockout platform in hPSCs.

We turned our attention to developing an inducible knockout approach. Current inducible CRISPR/Cas9 methods rely on conditional overexpression of Ca59 in the presence of a constitutively expressed gRNA. In this case, control of Ca59 overexpression is achieved by a TET-ON method in which following doxycycline treatment a tetracycline—controlled reverse transactivator (rtTA) activates a Pol ll-dependent tetracycline responsive element (TRE) er (a fusion between multiple TET s and a minimal CMV promoter). While this TET—ON platform has been successfully applied to certain human cell types, we observed that this inducible system is silenced during hPSC differentiation into multiple lineages (including cardiomyocytes, hepatocytes, and smooth muscle , even after targeting into the AAVSl GSH (data not shown). We explored the possibility to develop an alternative and improved method by combining a constitutively sed CAG promoter-driven Ca59 with an inducible gRNA cassette based on the one developed for inducible shRNA expression (Fig. 18A,B). We therefore ted hESCs lines in which a scent reporter gene could be knocked out in an inducible fashion (Fig. 18C). For this, we targeted ROSAZG—EGFPdZ reporter hESCs with both an inducible EGFP gRNA and a tutive Ca59 in the AAVSl locus, each transgene being integrated into one of the two alleles. This dual targeting approach was rapid (<2 weeks) and efficient (>90% of lines ning both transgenes. Remarkably, when individual clonal sublines were grown in the presence of tetracycline we observed decreased EGFPd2 sion in all of the targeted lines, and EGFPd2 homozygous cells showed near—homogeneous loss of at least one copy of the reporter gene as early as five days following tetracycline induction (as demonstrated by 50% reduction in EGFPd2 fluorescence). Prolonged ent with tetracycline led to progressive full loss of EGFPd2 fluorescence in up to 75% EGFPd2 homozygous cells (data not .

Interestingly, ression of either two or three copies of the same EGFP gRNA cassette from the same AAVSl locus was ient to significantly increase the speed and efficiency of inducible EGFPd2 knockout in all the clonal sublines analysed. For instance, simultaneous induction of three copies of the same gRNA resulted in a remarkable 95% knockout ency following tetracycline treatment. Importantly, inducible EGFPd2 knockout hESCs did not show any icant decrease neither in the proportion of EGFPd2 positive cells nor in their fluorescence after prolonged culture in the absence of tetracycline, even when several gRNA copies were used. This demonstrated that the inducible gRNA expression was tightly controlled. Finally, testing of additional gRNAs against EGFPd2 revealed that the speed and efficiency of inducible knockout strongly relied on the gRNA. Indeed, an optimal sequence allowed up to 90% knockout after only 2 days of induction. Of note, the most efficient gRNA also resulted in uncontrolled EGFPd2 knockout, but this tion was avoided by simply adding a second TET operon to the inducible H1 promoter to ensure even more stringent transcriptional control. Collectively, these results show that the knockdown system could be y repurposed to support inducible gRNA expression and allow tightly—controlled activity of CRISPR/Cas9 over a broad range of gRNA potency. To the best of our knowledge, this is the first conditional CRISPR/Cas9 approach based on inducible gRNA expression.

Claims (15)

The claims defining the invention are as follows:

1. An ex vivo method for controlling transcription of a c sequence in a cell comprising: a) targeted insertion of a gene encoding a riptional regulator protein into a first genetic safe harbour site; and b) targeted insertion of an inducible cassette into a second genetic safe harbour site; wherein said ble cassette comprises said genetic ce ly linked to an inducible promoter, and said promoter is regulated by the transcriptional regulator protein; wherein said first and second genetic safe harbour sites are different; and wherein the cell is an animal cell.

2. An ex vivo method for the production of myocytes from pluripotent stem cells, comprising the steps a) targeted insertion of a gene encoding a transcriptional regulator protein into a first c safe harbour site; and b) targeted insertion of the MYOD1 gene operably linked to an inducible promoter into a second genetic safe r site; and c) culturing said cells in the presence of retinoic acid; wherein said inducible promoter is regulated by the transcriptional regulator protein; wherein said first and second genetic safe harbour sites are different; and wherein the pluripotent stem cell is an animal cell.

3. An ex vivo method according to claim 1 or 2, wherein the cell is a mammalian, marsupial, non-human primate, d or livestock animal cell, or ably the cell is from a livestock animal.

4. An ex vivo method according to any one of the preceding claims, wherein the cell is from a pig or from cattle.

5. An ex vivo method according to any one of the preceding claims, wherein the method is for the programming of pluripotent stem cells into mature cells.

6. An ex vivo method according to any one of the ing claims, wherein said transcriptional regulator protein is selected from any one of: reverse tetracycline transactivator protein (rtTa), Tetracycline sor (TetR), VgEcR synthetic receptor, or a hybrid transcriptional regulator protein comprising a DNA binding domain from the yeast GAL4 protein, a truncated ligand binding domain from the human progesterone receptor and an activation domain from the human NF-κB.

7. An ex vivo method according to claim 6, wherein the activity of rtTA is controlled by tetracycline or a derivative thereof, optionally doxycycline, and wherein the inducible promoter includes a Tet Responsive Element (TRE).

8. An ex vivo method ing to any one of the preceding claims, wherein said first and second c safe harbour sites are selected from any two of the hROSA26 locus, the AAVS1 locus, the CLYBL gene or the CCR5 gene.

9. An ex vivo method according to any one of the preceding claims, wherein additional genetic material is inserted at the first and/or second genomic safe harbour sites, optionally comprising one or more of the a) suicide gene; b) selectable marker; c) reporter gene; and/or d) gene for a non-coding RNA.

10. A cell obtainable by the method according to any one of the preceding claims.

11. An animal cell with a modified genome that comprises a gene encoding a transcriptional tor protein inserted into a first c safe harbour site; and an inducible cassette comprising a genetic sequence ly linked to an inducible promoter inserted into a second genetic safe r site; wherein said inducible promoter is regulated by the transcriptional regulator protein and said first and second genetic safe harbour sites are different.

12. An animal cell according to claim 11, n the ble cassette comprises the genetic sequence encoding MYOD1.

13. Ex vivo or in vitro use of a cell according to any one of claims 10-12 for tissue engineering, optionally for the production of cultured meat.

14. An ex vivo method for reducing the transcription and/or translation of an nous gene in an animal cell, comprising the following steps: a) targeted insertion of a gene encoding a transcriptional regulator n into a first genetic safe harbour site; and b) targeted insertion of an inducible cassette comprising DNA ng a non-coding RNA sequence operably linked to an inducible promoter into a second genetic safe harbour site; wherein said promoter is regulated by the transcriptional regulator protein; n said non-coding RNA sequence suppresses the transcription or translation of an endogenous gene; wherein said first and second genetic safe harbour sites are different.

15. An ex vivo method for knocking out an endogenous gene in an animal cell, comprising the following steps: a) targeted insertion of a gene encoding a transcriptional regulator n and a gene encoding Cas9 into a first genetic safe harbour site; and b) targeted insertion of an inducible te comprising a guide RNA operably linked to an inducible promoter into a second genetic safe harbour site; wherein said promoter is regulated by the transcriptional tor protein; wherein said gRNA sequence targets the endogenous gene; and wherein said first and second genetic safe harbour sites are different. mm m0» mowmcmx Eamfiéa wn NO» 5.3 mu magma“ 383?: i @38me b3 n on mass v83. man GO» mamozgéa ”.0“, oargx mm, om mm a? mm C.) if) a Li) N X8W m, mm, m (xew :0 %) yaw \Rwfim «3 me” EQHQEV m. 3“%% ”S anew 60 VFW gmwmw§§ 50%;. “Kg.“ 23m an; am mm we .8 § 58m 1“,XDum IX:0 % } ma: m Q'PTi~OX hPSCS C?" Neuronat Games M 0) NM NGNE \\\\\\\' BRN2 sion hPSCs M (go \\\\\\\\\\\\\