A Method of Altering a Differentiation Status of a Cell

Abstract

The invention relates to a method of altering a differentiation status of a stem cell by modulating the expression of one or more differentiation factors with a nuclease-deactivated Cas9 (dCas9) fusion protein comprising dCas9 and a transcriptional activator. The method may further include a guide RNA (gRNA) and an activator module comprising RNA-binding protein binding capable of binding to the gRNA. In one embodiment, the dCas9 fusion protein comprises dCas9 and VP64 while the activator module comprises MS2 coat protein and p65. The one or more differentiation factors may comprise PAX6, MITF and OTX2 for differentiation of pluripotent stem cell into retinal pigment epithelium (RPE). Also disclosed are cells comprising the dCas9 fusion protein, gRNA, kits, and method of treating a disease thereof.

Claims

1. A method of altering a differentiation status of a cell, the method comprising: modulating the expression of one or more differentiation factors with a nuclease-deactivated Cas9 (dCas9) fusion protein, the dCas9 fusion protein comprising dCas9 and an effector comprising a transcriptional regulator, optionally the transcription regulator is a transcriptional activator.

2. The method of claim 1, the method further comprising: providing a guide RNA (gRNA) in the cell, wherein the gRNA is capable of guiding the dCas9 fusion protein to a target site that is/that is in proximity of a promoter region of the one or more differentiation factors to allow the dCas9 fusion protein to modulate the expression of the one or more differentiation factors, optionally wherein the target site that is/that is in proximity of the promoter region is within an about −300 base pairs (bp) to about +5 bp window of the promoter region.

3. (canceled)

4. The method of claim 1, the method further comprising: providing an activator module comprising a RNA-binding protein capable of binding to the gRNA, optionally wherein the RNA-binding protein comprises MS2 coat protein (MCP), optionally wherein the activator module further comprises one or more transcriptional activators, optionally the transcriptional activator is selected from the group consisting of VP64, p65, HSF1, Rta and combinations thereof, optionally wherein the activator module comprises p65 and/or HSF1.

5.-6. (canceled)

7. The method of claim 1, wherein the dCas9 fusion protein comprises VP64 and optionally, p65 and/or Rta, or the method further comprising expressing the dCas9 fusion protein, optionally a dCas9-VP64 fusion protein and/or a dCas9-VP64-p65-Rta (dCas9-VPR) fusion protein, prior to the modulating step, or the method comprises modulating the expression of one or more differentiation factors with a CRISPR/dCas9 synergistic activation mediators (CRISPR/dCas9-SAM) complex/dCas9 ribonucleoprotein complex (e.g. a complex comprising the dCas9 fusion protein)/dCas9-VP64/dCas9-VPR/dCas9-VP64 and MS2-P65-HSF1.

8.-9. (canceled)

10. The method of claim 1, wherein the one or more differentiation factors comprises transcription factors, optionally wherein the cell is a stem cell, stem cell-like cell, a progenitor cell or a precursor cell, optionally the cell comprises one that is selected from the group consisting of embryonic stem cell (e.g. hESC3), adult stem cell, induced pluripotent stem cell (iPSC), mesenchymal stem cell (MSC), human embryonic kidney cell (HEK293) and the like.

11. (canceled)

12. The method of claim 1, wherein the method is a method of differentiating a cell, optionally the one or more differentiation factors influence an expression of a neuroprogenitor gene and/or a retinal pigment epithelium (RPE)-associated gene, optionally the RPE-associated gene comprises a gene associated with a mature RPE/RPE specific mature gene, a gene associated with pigmentation/RPE specific pigmentation gene or early eye field gene.

13. (canceled)

14. The method of claim 1, wherein the one or more differentiation factors is selected from the group consisting of PAX6, MITF, OTX2 and combinations thereof, optionally the one or more differentiation factors is selected from the group consisting of LHX2, RAX2, Tyrosinase, CRALBP, BEST1, RPE65, PEDF, pme117, PYR, Trypl, Tryp2, CRX and combinations thereof.

15. (canceled)

16. The method of claim 1, wherein the cell produced from the method expresses premelanosome marker 17 (PMEL17), optionally the expression of PMEL17 in the produced cell is at least about 50%, or wherein the cell produced from the method expresses Pax6, optionally the cell is a neuroprogenitor cell.

17.-19. (canceled)

20. The method of claim 1, wherein the method is free of modulating the expression of a transcription activator selected from the group consisting of: cMyc, Klf4, Nrl, Crx, Rax, LHX2, SIX3, SOX9, GLIS3, FOXD1, ZNF92 , C11or19 and combinations thereof directly via the dCas9 fusion protein, or the method is free of the use of a gRNA specific to a target site that is/that is in proximity of a promoter region of: cMyc, Klf4, Nrl, Crx, Rax, LHX2, SIX3, SOX9, GLIS3, FOXD1, ZNF92, C11orf9 and combinations thereof, or the method is free of exogenous growth factor, free of inducible system, and/or is free of whole exogenous nucleic acid, optionally wherein modulating the expression of one or more differentiation factors comprises an endogenous activation of the one or more differentiation factors.

21.-27. (canceled)

28. A guide RNA (gRNA) to a target site that is or that is in proximity of the promoter region of one or more differentiation factors to modulate the expression of the one or more differentiation factors, wherein the gRNA is configured to guide a fusion protein selected from the group consisting of dCas9 fusion protein, CRISPR/dCas9 synergistic activation mediators (CRISPR/dCas9-SAM) complex, dCas9 ribonucleoprotein complex, dCas9-VP64, dCas9-VPR, dCas9-VP64, and MS2-P65-HSF1, optionally wherein the gRNA is a single/short gRNA (sgRNA).

29. The gRNA of claim 28, wherein at least a portion of the guide RNA is capable of binding to the target site/target genomic locus that is in an about −300 base pairs (bp) to about +5 bp window of the promoter region of one or more differentiation factors selected from the group consisting of PAX6, MITF, OTX2, and combinations thereof.

30. The gRNA of claim 28, wherein the gRNA has about 15 bp to about 25 bp, optionally wherein the gRNA has at least about 80% identity with a sequence selected the group consisting of SEQ ID NO: 1 (AATGTGTGTGTGCCGGCGCC), SEQ ID NO: 2 (GCCAGCACACCTATGCTGAT), SEQ ID NO: 3 (GCTTCGCTAATGGGCCAGTG), SEQ ID NO: 4 (ACAATAAAATGGGCTGTCAG), SEQ ID NO: 5 (GAGTGAGAGATAAAGAGTGT), SEQ ID NO: 6 (CGGGCCGAACTACAGATCCC), SEQ ID NO: 7 (CCAAACAGGAGTTGCACTAG), SEQ ID NO: 8 (AGCTGTAGTTTTCGTGGGAG), SEQ ID NO: 9 (GCGGGGGAGAGGCAACGTGG), SEQ ID NO: 10 (CTGTACCCTTGAAGCAAGTG), SEQ ID NO: 11 (GAACATTCTGGTAATGTCGG), SEQ ID NO: 12 (GCGTCAAAAAGTTGCCAGAG), SEQ ID NO: 13 (AACAGGCCGCTGCTGCACGG), SEQ ID NO: 14 (GATTGACACATCTAAGCCAG), SEQ ID NO: 15 (TAAAAACACACAACAGGGGG), SEQ ID NO: 76 (GGGGTGGCCCAGGGACTCTG), SEQ ID NO: 77 (TGTGCGTGAGGGGTCGCCAG), SEQ ID NO: 78 (GCCCCTGCTCTGACCCCGGG), SEQ ID NO: 79 (GGAGAGGCTGTGTGCGTGAG), SEQ ID NO: 80 (GAACTGTATAAAAGCGCCGG), SEQ ID NO: 81 (CCTAATCTGCCAAACTTCTG), SEQ ID NO: 82 (GAGGCGTGTCCGGAGCAGGC), SEQ ID NO: 83 (GGTAGGCGAGAAGCAGGCAA), SEQ ID NO: 84 (TCCTTCCCTTCCGGAGCCCG), SEQ ID NO: 85 (GAGCCACCAGACACTGGTGA), SEQ ID NO: 86 (CCCTATCCAAATCTTCTCCG), SEQ ID NO: 87 (ACTTCTGCCCAATCAGAGAA), SEQ ID NO: 88 (AAGAGAAGGCGTCACTTCCG), SEQ ID NO: 89 (AGCAGGTCATACGCCTGCCT), SEQ ID NO: 90 (AAGAGCTCTTAAATACACAG), SEQ ID NO: 91 (GTGACCACAAAATGCCAGGG), SEQ ID NO: 92 (CGGGGGAACTACCTGAACTG), SEQ ID NO: 93 (GGCCCTTATCAGCCACACAT), SEQ ID NO: 94 (AGGCTCACCGTTCCCATGTG), SEQ ID NO: 95 (GTGTCCAAGACAATGCAGGG), SEQ ID NO: 96 (GGGCAAGGCGACGTCAAAGG), SEQ ID NO: 97 (GCGAAAGTTTTGTGAAATTG), SEQ ID NO: 98 (GGGGGGCAAGGCGACGTCAA), and SEQ ID NO: 99 (CACCAAATTTGCATAAATCC).

31.-32. (canceled)

33. The gRNA of claim 28, wherein the gRNA is provided in a set comprising at least two of the gRNA, wherein the gRNA is selected from the group consisting of: a gRNA that is specific to a target site that is/that is in proximity of the promoter region of PAX6, a gRNA that is specific to a target site that is/that is in proximity of the promoter region of MITF and a gRNA that is specific to a target site that is/that is in proximity of the promoter region of OTX2.

34. The gRNA of claim 28, wherein the gRNA is cloned with a oligonucleotide/primer having at least about 80% with a sequence selected from Table 2 below: TABLE-US-00009 TABLE 2 SEQ ID Name Sequence NO. Pax6_1_Fwd CACCGACAATAAAATGGGCTGTCAG 16 Pax6_1_Rev AAACCTGACAGCCCATTTTATTGTC 17 Pax6_2_Fwd CACCGGAGTGAGAGATAAAGAGTGT 18 Pax6_2_Rev AAACACACTCTTTATCTCTCACTCC 19 Pax6_3_Fwd CACCGGCCAGCACACCTATGCTGAT 20 Pax6_3_Rev AAACATCAGCATAGGTGTGCTGGCC 21 Pax6_4_Fwd CACCGAATGTGTGTGTGCCGGCGCC 22 Pax6_4_Rev AAACGGCGCCGGCACACACACATTC 23 Pax6_5_Fwd CACCGGCTTCGCTAATGGGCCAGTG 24 Pax6_5_Rev AAACCACTGGCCCATTAGCGAAGCC 25 MITF_1_Fwd CACCGCGGGCCGAACTACAGATCCC 26 MITF_1_Rev AAACGGGATCTGTAGTTCGGCCCGC 27 MITF_2_Fwd CACCGCCAAACAGGAGTTGCACTAG 28 MITF_2_Rev AAACCTAGTGCAACTCCTGTTTGGC 29 MITF_3_Fwd CACCGGCGGGGGAGAGGCAACGTGG 30 MITF_3_Rev AAACCCACGTTGCCTCTCCCCCGCC 31 MITF_4_Fwd CACCGAGCTGTAGTTTTCGTGGGAG 32 MITF_4_Rev AAACCTCCCACGAAAACTACAGCTC 33 MITF_5_Fwd CACCGCTGTACCCTTGAAGCAAGTG 34 MITF_5_Rev AAACCACTTGCTTCAAGGGTACAGC 35 OTX2_1_Fwd CACCGGCGTCAAAAAGTTGCCAGAG 36 OTX2_1_Rev AAACCTCTGGCAACTTTTTGACGCC 37 OTX2_2_Fwd CACCGGAACATTCTGGTAATGTCGG 38 OTX2_2_Rev AAACCCGACATTACCAGAATGTTCC 39 OTX2_3_Fwd CACCGTAAAAACACACAACAGGGGG 40 OTX2_3_Rev AAACCCCCCTGTTGTGTGTTTTTAC 41 OTX2_4_Fwd CACCGAACAGGCCGCTGCTGCACGG 42 OTX2_4_Rev AAACCCGTGCAGCAGCGGCCTGTTC 43 OTX2_5_Fwd CACCGGATTGACACATCTAAGCCAG 44 OTX2_5_Rev AAACCTGGCTTAGATGTGTCAATCC 45

35. The gRNA of claim 28, comprised in a composition comprising: a dCas9 fusion protein, the dCas9 fusion protein comprising dCas9 and an effector; and optionally an activator module comprising a RNA-binding protein capable of binding to the gRNA, further optionally wherein the RNA-binding protein comprises MS2 coat protein (MCP).

36.-37. (canceled)

38. A method of treating a disease, the method comprising transplanting, to a patient in need thereof, (i) a cell comprising a dCas9 fusion protein that is configured to modulate the expression of one or more differentiation factors, the dCas9 fusion protein comprising dCas9 and an effector, or progenies thereof, or (ii) a cell that has a second differentiation status (or its progenies thereof) that was differentiated from a cell having a first differentiation status.

39. The method of claim 38, wherein the disease is an eye disease/disorder, optionally wherein the eye disease/disorder is selected from the group consisting of macular degeneration, acute macular degeneration (AMD), atrophic age-related macular degeneration (atrophic AMD), dry age-related macular degeneration (Dry-type AMD), retinitis pigmentosa (RP), Stargardt's disease, and myopia.

40. The method of claim 38, wherein the cell in (i) comprises a guide RNA (gRNA) capable of guiding the dCas9 fusion protein to a target site that is/that is in proximity of the promoter region of the one or more differentiation factors to allow the dCas9 fusion protein to modulate the expression of the one or more differentiation factors.

41. The method of claim 38, wherein the cell in (ii) has the second differentiation status is devoid of a dCas9 fusion protein or a CRISPR/dCas9-SAM complex.

Description

DETAILED DESCRIPTION OF FIGURES

[0219] Example embodiments of the disclosure will be better understood and readily apparent to one of ordinary skill in the art from the following discussions and if applicable, in conjunction with the figures. It should be appreciated that other modifications related to structural, electrical and optical changes may be made without deviating from the scope of the invention. Example embodiments are not necessarily mutually exclusive as some may be combined with one or more embodiments to form new exemplary embodiments.

[0220] FIG. 1. CRISPR/dCas9-SAM expressing stable cell generation. (a) Schematic representation of CRISPR/dCas9-SAM structures. (b) Schematic diagram of the experimental procedure used for generating CRISPR/dCas9-SAM expressing stable human pluripotent cells.

[0221] FIG. 2. Directed differentiation approach from pluripotent stem cells to RPEs. (a) Schematic diagram of RPE transcription factors activation network of important RPE specific genes. (b) Schematic diagram of the hypothesis of the present disclosure, by delivering guide RNA sequences specific to activate the three main transcription factors (PAX6, MITF and OTX2) simultaneously in pluripotent stem cells expressing CRISPR/dCas9-SAM may differentiate directly into RPE cells.

[0222] FIG. 3. Functional screening of designed guide RNAs to endogenously activate (a) PAX6, (b) MITF and (c) OTX2 in CRISPR/dCas9-SAM pluripotent stem cells. CRISPR/dCas9-SAM pluripotent stem cells were transduced with indicated sgRNA lentivirus supernatants for each gene of interest. qRT-PCR analysis of mRNA expression levels were measured 4 days post transduction. The mRNA expression levels were normalized by GAPDH and then standardized to that in the sample of sgControl. Values shown are the mean±SE of n=3.

[0223] FIG. 4. Endogenous gene activation by using concentrated lentivirus supernatants individually in CRISPR/dCas9-SAM pluripotent stem cells. (a) Schematic diagram of the transduction procedure using concentrated individual top three guides based on our previous gRNA screening. (b) qRT-PCR analysis of mRNA expression levels were measured 4 days post transduction. The mRNA expression levels were normalized by GAPDH and then standardized to that in the sample of sgControl. Values shown are the mean±SE of n=3.

[0224] FIG. 5. Multiplex gene activation using concentrated lentivirus supernatants in CRISPR/dCas9-SAM pluripotent stem cells. (a) Schematic diagram of the transduction procedure for multiplex gene activation using concentrated top guide RNA for each gene. (b) qRT-PCR analysis of mRNA expression levels were measured 4 days post transduction. The mRNA expression levels were normalized by GAPDH and then standardized to that in the sample of sgControl. Values shown are the mean±SE of n=3.

[0225] FIG. 6. hiPSC RPE differentiation using CRISPR/dCas9-SAM mediated multiplex endogenous gene activation. (a) Schematic of hiPSC differentiation into RPE using CRISPR/dCas9-mediated multiplex gene activation followed by culture in RPEM. (b) qRT-PCR analysis of pluripotency (Oct4), activated transcription factors (PAX6 (+5a), MITF and OTX2), early eye field genes (LHX2 and RAX), RPE specific pigmentation genes (Tyrosinase, pMEL17, TYRP1 and TYRP2) and RPE specific mature genes (CRALBP, RPE65, BEST1 and PEDF). (c) Flow cytometry histograms of PAX6 and pMEL17 at day 18, 24 and 40. (d & e) Morphology of differentiating hiPSC at day40 in RPEM and RPE cells appear as distinct pigmented foci, with (d) showing macroscopic and (b) showing microscopic images. (Scale bar: 200 μm)

[0226] FIG. 7 Characterization of cells during hiPSC RPE differentiation using CRISPR/dCas9-SAM mediated multiplex endogenous gene activation. (a) Comparison of RPE specific gene expression from hRPE (Lonza) and day 28 of hiPSC-CRISPR/dCas9-SAM activated RPE differentiation. (b) Comparison of hiPSC-CRISPR/dCas9-SAM activated RPE differentiation on Matrigel (Gtx) and Laminin 521 (Ln521) following days 4, 10 and 16 of gene activation

[0227] FIG. 8. Lentiviral RPE triple sgRNA vector design and characterization. (a) Lentiviral plasmid encoded with PAX6, MITF and OTX2 sgRNA sequences. Each sgRNA with MS2 scaffold is placed under the control of U6 promoter followed by a terminator sequence. (b) Gene expression data from iPSC CRISPR-SAM cells transduced with triple guide lentivirus at different concentration. The error bars represent the standard error of the mean. Abbreviations: PAX6 (Paired box protein), OTX2 (Orthodenticle homeobox 2), MITF (Melanocyte Inducing Transcription Factor).

[0228] FIG. 9 Characterization of hiPSC RPE differentiation using triple guide lentivirus and the mix of individual, Pax6 (P), MITF (M) and OTX2 (0) lentiviruses. (a) Schematic representation of the experiment design. (b) Progression of gene expression profile of pluripotency marker (Oct4-), RPE differentiation early and mature markers. iPSC-CRISPR SAM cells were plated as single cells (as before) on day 0, qPCR data on days 4, 10 and 18. The error bars represent the standard error of the mean. Abbreviations: Oct-4 (Octamer-binding transcription factor 4), PAX6 (Paired box protein), OTX2 (Orthodenticle homeobox 2), MITF (Melanocyte Inducing Transcription Factor), pMEL17 (Melanocyte protein), TyrP1 (Tyrosinase Related Protein-1), CRALBP (Retinaldehyde-binding protein-1), RPE65 (Retinoid isomerohydrolase), BEST1 (Bestrophin-1), PEDF (Pigment epithelium-derived factor), TyrP2 (Tyrosinase Related Protein-2), LHX2 (LIM Homeobox 2), RAX (Retinal homeobox protein).

[0229] FIG. 10 Characterization of hiPSC RPE differentiation using triple guide lentivirus. (a) Time-course of gene expression progression of RPE markers. The error bars represent the standard error of the mean. (b) FACS analysis of the expression of RPE specific markers (% positive cells) during differentiation. (c) Pigmentation and cobble stone morphology of RPE cells are shown as phase contrast images. Abbreviations: Oct-4 (Octamer-binding transcription factor 4), PAX6 (Paired box protein), OTX2 (Orthodenticle homeobox 2), MITF (Melanocyte Inducing Transcription Factor), pMEL17 (Melanocyte protein), TyrP1 (Tyrosinase Related Protein-1), CRALBP (Retinaldehyde-binding protein-1), RPE65 (Retinoid isomerohydrolase), BEST1 (Bestrophin-1), PEDF (Pigment epithelium-derived factor), TyrP2 (Tyrosinase Related Protein-2), LHX2 (LIM Homeobox 2), RAX (Retinal homeobox protein), ZO-1 (Zonula occludens-1).

[0230] FIG. 11 Characterization of iPSC-dCas9 SAM cells differentiation into RPE cells using triple sgRNA lentivirus. (a) Schematic representation of the protocol and timeline for RPE differentiation. On day 18, the RPE progenitor cells were split and tested individually under three different conditions (5% FBS, No FBS and 5% KOSR). qPCR analysis comparing the time-course of eye field, early and mature RPE genes during RPE differentiation (b) days 4-18 after RPE triple virus transduction & (c) days 7-21 after the cells were split on day 18 under three different media conditions. The error bars represent the standard error of the mean. (d) Phase contrast images of iPSC-dCas9 SAM cells differentiated into RPE cells (pigmentation and cobble stone morphology) 21 days (p1) after replating on Dayl8 in 5% KOSR media. (e) Immunofluorescence images of mature RPE-specific tight junction markers (Bestrophin-1, CRALBP, N-Cadherin, Occludin & ZO-1), pigmentation marker (PMEL17) and CRISPR-activated RPE transcription factors (Pax6, Mitf & Otx2) from cells grown in 5% KOSR. Scale bar=100 μm. (f) Representation of flow cytometry histograms for RPE pigmentation marker (PMEL17), CRISPR-activated TFs (Pax6, Otx2, MITF) and pluripotency markers (Oct4- and TRA-1-60) at day 40 in 5% KOSR. Abbreviations: Oct-4 (Octamer-binding transcription factor 4), PAX6 (Paired box protein), OTX2 (Orthodenticle homeobox 2), MITF (Melanocyte Inducing Transcription Factor), pMEL17 (Melanocyte protein), TyrP1 (Tyrosinase Related Protein-1), CRALBP (Retinaldehyde-binding protein-1), RPE65 (Retinoid isomerohydrolase), BEST1 (Bestrophin-1), PEDF (Pigment epithelium-derived factor), TyrP2 (Tyrosinase Related Protein-2), LHX2 (LIM Homeobox 2), RAX (Retinal homeobox protein), ZO-1 (Zonula occludens-1).

[0231] FIG. 12 Characterization of hESC-dCas9 SAM cells differentiation into RPE cells using triple sgRNA lentivirus. The protocol for iPSC-dCas9 SAM cells shown in FIG. 4 was validated using hESC-dCas9 SAM cells. qPCR analysis comparing the time-course of eye field, early and mature RPE genes during RPE differentiation (a) days 4-17 after RPE triple virus transduction & (b) day 11 after the cells were split on day 18 in 5% KOSR. For the control, cells (without virus transduction) were maintained in 5% FBS and after day 17 the cells were split and maintained in 5% FBS (5% FBS_ctrl) and 5% KOSR (5% FBS to 5% KOSR_ctrl). The error bars represent the standard error of the mean. (c) Phase contrast images of hESC-dCas9 SAM cells differentiated into RPE cells (pigmentation and cobble stone morphology) 21 days (p1) after replating on Dayl7. Abbreviations: Oct-4 (Octamer-binding transcription factor 4), PAX6 (Paired box protein), OTX2 (Orthodenticle homeobox 2), MITF (Melanocyte Inducing Transcription Factor), pMEL17 (Melanocyte protein), TyrP1 (Tyrosinase Related Protein-1), CRALBP (Retinaldehyde-binding protein-1), RPE65 (Retinoid isomerohydrolase), BEST1 (Bestrophin-1), PEDF (Pigment epithelium-derived factor), TyrP2 (Tyrosinase Related Protein-2), LHX2 (LIM Homeobox 2), RAX (Retinal homeobox protein), ZO-1 (Zonula occludens-1).

[0232] FIG. 13 Characterization of iPSC-dCas9 SAM cells differentiation into RPE using triple sgRNA lentivirus or MITF sgRNA lentivirus only. (a) Schematic representation of the protocol used for this study. (b) Time-course qPCR analysis showing progression of RPE differentiation from triple sgRNA lentivirus transduced cells, wherein the control (no virus) and MITF sgRNA lentivirus transduced cells have very low RPE markers expression. The error bars represent the standard error of the mean. Note triple sgRNA transduced cells shows higher RPE marker expression as compared to MITF sgRNA only and control cells. (c) Morphology of iPSC-dCas9 SAM cells after 39 days of differentiation. Note the absence of pigmented clusters in the control and MITF sgRNA lentivirus transduced wells. (d) Phase contrast microscopic images of iPSC-dCas9 SAM cells differentiated into RPE using triple sgRNA guide displaying the pigmented and cobblestone morphology on day 39. Scale bar: 100 μm. (e) Representative flow cytometry histograms of RPE and pluripotency markers expression on day 18 (P0) and day 39 (P1) cells transduced with triple sgRNA lentivirus. Abbreviations: Oct-4 (Octamer-binding transcription factor 4), PAX6 (Paired box protein), OTX2 (Orthodenticle homeobox 2), MITF (Melanocyte Inducing Transcription Factor), pMEL17 (Melanocyte protein), TyrP1 (Tyrosinase Related Protein-1), CRALBP (Retinaldehyde-binding protein-1), RPE65 (Retinoid isomerohydrolase), BEST1 (Bestrophin-1), PEDF (Pigment epithelium-derived factor), TyrP2 (Tyrosinase Related Protein-2), LHX2 (LIM Homeobox 2), RAX (Retinal homeobox protein).

[0233] FIG. 14 Endogenous activation of erythropoietin (EPO) growth factor in human iPSC-dCas9 SAM cells. (a) qPCR data collected after 4 days of EPO sgRNAs lentivirus (unconcentrated) transduction. (b) EPO ELISA was carried out using two commercially available sources to quantify the CRISPRa EPO. For this, the spent mTesR medium of cells transduced with EPO_g2 were collected on days 3 and 4, the pooled medium was concentrated using Amicon Ultra-15 centrifugal filter (10 KDa cutoff membrane) since the EPO MW is 21 KDa. The retentate was used for quantification using ELISA. According to the standards, the EPO secreted in the medium is in the range of 47-51 IU/mL of EPO.

[0234] FIG. 15 Endogenous activation of growth factors in human iPSC-dCas9 SAM cells. qPCR data analysis of stem cell factor (SCF), thrombopoietin (TPO), granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte-colony stimulating factor (G-CSF).

[0235] FIG. 16 Endogenous activation of factors in HEK293_CRISPR dCas9 SAM cells. qPCR data analysis of (a) erythropoietin (EPO) and (b) stem cell factor (SCF) genes.

EXAMPLES

[0236] Materials and Method

[0237] Establishment of Stable Cell Line:

[0238] To establish stably expressing CRISPR/dCas9-SAM expressing pluripotent stem cells, iPSC and hESC3 cells were plated on GelTrex-coated 96-well tissue culture plate at approximately 2.4×10.sup.4 cells in 110 μL of mTesR1 medium with 10 μM ROCK inhibitor. After 24 h, cells were transduced with the lentiviral vectors (FIG. 1a), dCas9-VP64 (Addgene: 61425) and MS2-p65-HSF1 (Addgene: 61426) together at multiplicity of infection (MOI=2) with 8 μg/mL polybrene (hexadimethrine bromide. 24 h after transduction, the culture medium was replaced with mTesR1 containing the selection antibiotics (Hygromycin B, 50 μg/mL and Blasticidin S, 4 μg/mL). The mTesR1 medium with antibiotics was replaced every day for 4-7 days, until there are no viable cells in the no-virus control. iPSC-CRISPR dCas9 SAM and hESC-CRISPR dCas9 SAM cells, were passaged an additional two days with mTesR1 medium without antibiotics and were subsequently passaged and banked accordingly.

[0239] Guide RNA Design and Plasmid Construction:

[0240] Guide RNAs were designed and assembled as described by Konermann et al.sup.3. For each gene, 5 sgRNA target sites spread across the proximal promoter between −200 bp to +1 bp window were selected. The sgRNA sequences are listed in Table 1. Briefly, the lentiviral vectors with different sgRNA sequences for each gene were generated by oligo cloning using the BsmBI site of lenti sgRNA(MS2)_zeo backbone (Addgene: 61427). Primers were supplied by Integrated DNA Technologies, IDT (Singapore) and sequences were verified through Axil Scientific Pte Ltd. (1st BASE, Singapore). The primer sequences are listed in Table 2.

TABLE-US-00006 TABLE 1 List of sgRNA sequences designed for this study. Specificity Efficiency Name Position Strand Sequence PAM Score Score Distance PAX6_4 31811163 1 AATGTGTGTGTGCCGGCGCC CGG 85.71532 45.53779 130 PAX6_3 31811122 1 GCCAGCACACCTATGCTGAT TGG 80.41661 57.03529 89 PAX6_5 31811191 −1 GCTTCGCTAATGGGCCAGTG AGG 74.83166 67.60208 172 PAX6_1 31811054 1 ACAATAAAATGGGCTGTCAG CGG 59.43153 66.63383 21 PAX6_2 31811082 1 GAGTGAGAGATAAAGAGTGT GGG 51.10384 64.62191 49 MITF_1 69739390 1 CGGGCCGAACTACAGATCCC AGG 87.69096 59.49774 42 MITF_2 69739276 1 CCAAACAGGAGTTGCACTAG CGG 83.95155 58.15318 94 MITF_4 69739338 1 AGCTGTAGTTTTCGTGGGAG CGG 77.456 53.51254 156 MITF_3 69739291 −1 GCGGGGGAGAGGCAACGTGG TGG 69.70525 62.74833 127 MITF_5 69739214 1 CTGTACCCTTGAAGCAAGTG GGG 66.46342 65.68941 218 OTX2_2 56810650 −1 GAACATTCTGGTAATGTCGG AGG 88.91548 64.17876 65 OTX2_1 56810495 −1 GCGTCAAAAAGTTGCCAGAG AGG 76.19507 66.72045 15 OTX2_4 56810615 1 AACAGGCCGCTGCTGCACGG GGG 71.60974 62.94672 121 OTX2_5 56810559 1 GATTGACACATCTAAGCCAG AGG 69.85061 64.23791 170 OTX2_3 56810590 1 TAAAAACACACAACAGGGGG AGG 64.48448 55.39254 96

TABLE-US-00007 TABLE 2 List of Primer sequences used for sgRNA cloning protocol. Seq ID No. Name Sequence 16 Pax6_1_Fwd CACCGACAATAAAATGGGCTGTCAG 17 Pax6_1_Rev AAACCTGACAGCCCATTTTATTGTC 18 Pax6_2_Fwd CACCGGAGTGAGAGATAAAGAGTGT 19 Pax6_2_Rev AAACACACTCTTTATCTCTCACTCC 20 Pax6_3_Fwd CACCGGCCAGCACACCTATGCTGAT 21 Pax6_3_Rev AAACATCAGCATAGGTGTGCTGGCC 22 Pax6_4_Fwd CACCGAATGTGTGTGTGCCGGCGCC 23 Pax6_4_Rev AAACGGCGCCGGCACACACACATTC 24 Pax6_5_Fwd CACCGGCTTCGCTAATGGGCCAGTG 25 Pax6_5_Rev AAACCACTGGCCCATTAGCGAAGCC 26 MITF_1_Fwd CACCGCGGGCCGAACTACAGATCCC 27 MITF_1_Rev AAACGGGATCTGTAGTTCGGCCCGC 28 MITF_2_Fwd CACCGCCAAACAGGAGTTGCACTAG 29 MITF_2_Rev AAACCTAGTGCAACTCCTGTTTGGC 30 MITF_3_Fwd CACCGGCGGGGGAGAGGCAACGTGG 31 MITF_3_Rev AAACCCACGTTGCCTCTCCCCCGCC 32 MITF_4_Fwd CACCGAGCTGTAGTTTTCGTGGGAG 33 MITF_4_Rev AAACCTCCCACGAAAACTACAGCTC 34 MITF_5_Fwd CACCGCTGTACCCTTGAAGCAAGTG 35 MITF_5_Rev AAACCACTTGCTTCAAGGGTACAGC 36 OTX2_1_Fwd CACCGGCGTCAAAAAGTTGCCAGAG 37 OTX2_1_Rev AAACCTCTGGCAACTTTTTGACGCC 38 OTX2_2_Fwd CACCGGAACATTCTGGTAATGTCGG 39 OTX2_2_Rev AAACCCGACATTACCAGAATGTTCC 40 OTX2_3_Fwd CACCGTAAAAACACACAACAGGGGG 41 OTX2_3_Rev AAACCCCCCTGTTGTGTGTTTTTAC 42 OTX2_4_Fwd CACCGAACAGGCCGCTGCTGCACGG 43 OTX2_4_Rev AAACCCGTGCAGCAGCGGCCTGTTC 44 OTX2_5_Fwd CACCGGATTGACACATCTAAGCCAG 45 OTX2_5_Rev AAACCTGGCTTAGATGTGTCAATCC

[0241] Lentivirus Production:

[0242] HEK293T cells were cultured in D10 medium at 37° C. with 5% CO.sub.2 and was maintained according to the manufacturer's recommendation. D10 recipe: Dulbecco's modified Eagle's medium (DMEM), Fetal bovine serum, heat-inactivated (10%), Penicillin G (100 units/mL) and Streptomycin (100 μg/mL). Cells were seeded into T175 flasks 20-24 h at a density of 1.8×10.sup.7 cells per flask in a total volume of 37 mL of D10 medium. Transfection was carried out using Lipofectamine 3000 reagent according to manufacturer's recommendation. Briefly, Lipofectamine 3000 reagent, lentivirus packaging plasmids (pMD2.G (8 μg)+pMDLg/pRRE (8 μg)+pRSV-Rev (8 μg)) and lenti expression vector (15 μg) with P3000 reagent were diluted in Opti-MEMTM I medium and were incubated for 10 min in room temperature. The solution mix with 50% of the A10 media was added directly to the cells and after 4h the medium was replaced with fresh pre-warmed D10 medium. Virus supernatant was harvested twice at 48 h and 72 h post transfection, and then filtered with a 0.45 μm PVDF filter (Millipore).

[0243] sgRNA Screening for Endogenous Gene Activation:

[0244] iPSC-CRISPR dCas9 SAM were plated at approximately 1×10.sup.5 cells/well in GelTrex-coated 12-well plate containing 1 ml of mTesR1 medium with 10 μM ROCK inhibitor. After 24 h, media was replaced with 0.5 mL of fresh mTesR1 media and 0.5 mL of top four sgRNA lentivirus supernatants of each gene target were added independently in different wells with 8 μg/mL polybrene. Fresh mTesR1 media with selection antibiotic, Zeocin (10 μg/ml) was replaced 24 h after transduction with daily media replenishments. Four days after transduction, cells total RNA samples were extracted for quantitative PCR analysis using Direct-zol RNA Miniprep kit (Zymo Research, CA).

[0245] sgRNA Lentivirus Concentration:

[0246] Top three performing guides were chosen for each gene and the concentration of the viral supernatants was carried out using Lenti-X-concentrator (Clontech) as per manufacturer's protocol. Briefly, for each guide viral supernatants from three T175 flasks were pooled and then filtered with a 0.45 μm PVDF filter (Millipore). To one volume of Lenti-X-concentrator, 3 volumes of clarified lentivirus supernatant was added and incubated at 4° C. for 30-45 min. The sample were centrifuged at 1500×g for 45 min at 4° C. The pellet was dissolved in 2.5 ml of sterile PBS and was stored at −80° C. in single-use aliquots.

[0247] Endogenous Gene Activation Using Concentrated sgRNA Lentivirus:

[0248] iPSC-CRISPR dCas9 SAM were plated at approximately 2×10.sup.4 cells/well in GelTrex-coated 12-well plate containing 1 ml of mTesR1 medium with 10 μM ROCK inhibitor. After 24 h, media was replaced with 1 mL of fresh mTesR1 and 3 μl of crude concentrated sgRNA lentivirus of each gene target were added independently in different wells with 8 μg/mL polybrene. For simultaneous activation of three genes, the ratio of sgRNAs targeting each gene was 1:1:1. Fresh mTesR1 media with selection antibiotic, Zeocin (10 μg/ml) was replaced 24 h after transduction with daily media replenishments. Four days after transduction, cells total RNA samples were extracted for quantitative PCR analysis using Direct-zol RNA Miniprep kit (Zymo Research, CA).

[0249] RPE Induction Using Gene Activation:

[0250] iPSC-CRISPR dCas9 SAM were plated at approximately 1×10.sup.5 cells/well in GelTrex-coated 12-well plate containing 1 ml of mTesR1 medium with 10 μM ROCK inhibitor. After 24 h, media was replaced with 1 mL of fresh mTesR1 and 3 μl of crude concentrated top performing sgRNA lentivirus of each gene target were added together with 8 μg/mL polybrene. 24 h after transduction, the medium was changed to RPE maintenance medium (RPEM) for 4-6 weeks with medium change twice a week. Samples were collected at different time points for quantitative PCR and flow cytometry analysis.

[0251] Quantitative Real-Time Polymerase Chain Reaction:

[0252] cDNA was synthesized from 1 μg of RNA using the Maxima First Strand cDNA Synthesis Kit (ThermoFisher). Quantitative real-time polymerase chain reaction (qPCR) was carried out using QuantStudio 3 Real-Time PCR System (ThermoFisher). The samples were run in biological triplicates and expression levels normalized using the geometric mean of the “housekeeping” gene: glyceraldehyde phosphate dehydrogenase (GAPDH). The primer sequences are listed in Table 3.

TABLE-US-00008 TABLE 3 Primers for gene expression analysis used in this study. Forward Reverse Ref. Oct3/4 CAGTGCCCGAAACCCACAC GGAGACCCAGCAGCCTCAAA 4 Lhx2 CGTCCGTCTTAACTTCTGTGC AGGTTGGTAAGAGTCGTTTGT Rax TCCCAGGAGGCTTGGAGACCC CTCCCCAAGTCCTGAGCGTGC Otx2 ACTTCCGAGAGCCATAGAAGG TAAGCAGATTGGTTTGTCCAT Pax6(+5a) CTCGGTGGTGTCTTTGTCAAC ACTTTTGCATCTGCATGGGTC 5 Mitf CCCAGTTCATGCAACAGAGAG GCAGAGGGAAGGGTGGTG Tyrosinase  GTGTAGCCTTCTTCCAACTCAG GTTCCTCATTACCAAATAGCATCC Tyrp1 GATTCCACTCTAATAAGCCCAAA TTCCAAGCACTGAGCGACAT Tyrp2 CTCAGACCAACTTGGCTACAGCTA CAGCACAAAAAGACCAACCAAA pmel17 TGATGGCTGTGGTCCTTGC CAGTGACTGCTGCTATGTGG PEDF TATCACCTTAACCAGCCTTTCATC GGGTCCAGAATCTTGCCAATG Best1 TAGAACCATCAGCGCCGTC TGAGTGTAGTGTGTATGTTGG Cralbp CACGCTGCCCAAGTATGATG CCAGGACAGTTGAGGAGAGG RPE65 CCTGATTCATACCCATCAGAACCC CACCACACTCAGAACTACACCATC GAPDH AGCAAGAGCACAAGAGGAAGAG GAGCACAGGGTACTTTATTGATGG 6

[0253] Flow Cytometry:

[0254] The samples were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. The samples (1×10.sup.5 cells) were incubated with primary (pmell7 (DAKO, DKO.M063429), Pax6 (DSHB), Mitf (Abcam, ab122982) and Otx2 (Merck, SAB5300043)) or isotype control antibodies at 1:100 concentration for 30 minutes at room temperature. Primary and isotype control were labeled with fluorophore conjugated secondary antibodies and control cells were incubated with only the secondary antibody for 30 minutes at room temperature. The labeled samples were run on NovoCyte 2000 flow cytometer (ACEA, Biosciences, Inc.). Data analysis was performed using FlowJo V10 software. The positive percentage was based on a background level set at 1% positive expression in samples labeled with isotype control antibodies.

[0255] Triple sgRNA Design

[0256] In order to improve the RPE induction efficiency in iPSC-CRISPR dCas9 SAM cells, a single vector encoding all three Pax6, Mitf and Otx2 sgRNA sequences was designed. Each of the sgRNA sequence with the MS2 scaffold expression is driven using the U6 promoter upstream of the sgRNA sequence (FIG. 8). The lentiviral vector construction service was provided by Vector Biolabs, USA. The design of the lentiviral vector backbone was carried out using their custom web-based lentiviral vector design tool. The custom built triple sgRNA encoded lentiviral vector was used to produce concentrated lentiviral particles as described earlier.

[0257] The lentiviral vector backbone encodes geneticin as an antibiotic selection marker. A minimum inhibitory concentration (MIC) assay for geneticin using iPSC-CRISPR dCas9 SAM cells was carried out and the MIC of geneticin was found to be 100 μg/mL.

[0258] Endogenous Gene Activation Study Using Triple sgRNA Lentivirus

[0259] iPSC-CRISPR dCas9 SAM were plated at approximately 1×10.sup.5 cells/well in GelTrex-coated 12-well plate containing 1 mL of mTesR1 medium with 10 μM ROCK inhibitor. After 24 h, media was replaced with 1 mL of fresh mTesR1 and different concentrations of crude concentrated triple sgRNA lentivirus was added together with 8 μg/mL polybrene. Fresh mTesR1 media with selection antibiotic, Geneticin (100 μg/ml) was replaced 24 h after transduction with daily media replenishments. Four days after transduction, cells total RNA samples were extracted for quantitative PCR analysis using Direct-zol RNA Miniprep kit (Zymo Research, CA).

[0260] RPE Induction Using Triple sgRNA Lentivirus

[0261] iPSC-CRISPR dCas9 SAM/hESC-CRISPR dCas9 SAM were plated at approximately 1×10.sup.5 cells/well in Laminin 521-coated 12-well plate containing 1 mL of mTesR1 medium with 10 μM ROCK inhibitor. After 24 h, media was replaced with 1 mL of fresh mTesR1 and 9 μL of crude concentrated triple sgRNA lentivirus was added together with 8 μg/mL polybrene. 24 h after transduction, the medium was changed to RPE maintenance medium (RPEM) for 2 weeks with medium change twice a week. On day 17/18 after transduction, the cells (RPE progenitors) were passaged and re-plated at 4×10.sup.5 cells/well in Laminin 521-coated 12-well plate containing RPE maintenance medium (RPEM) for 3 weeks with medium change twice a week. Samples were collected at different time points for quantitative PCR and flow cytometry analysis.

[0262] Immunofluorescence Assay

[0263] Cells were fixed with 4% paraformaldehyde in PBS and were blocked and permeabilized for 30 min in 10% goat serum and 2 ml of 0.1% Triton X-100 in PBS, and then incubated overnight at 4° C. with the following primary antibodies at 1:100 concentration: mouse anti-Occludin (Thermo Fisher, 331500), mouse anti-ZO-1 (Thermo Fisher, 339100), mouse anti-BEST1 (Abcam, ab2182), mouse anti-N-Cadherin (Novus Biologicals, NBP1-48309), mouse anti-CRALBP (Abcam, ab15051), mouse anti-MITF (abcam, ab3201), anti-PAX6 (DSHB), anti-OTX2 (Merck, SAB5300043) and mouse anti-PMEL17 (DAKO, DKO.M063429). Cells were then incubated for 1 h at room temperature with the corresponding secondary antibody conjugated to Alexa-488 (Invitrogen) and counterstained with Hoechst 33342 (Invitrogen). Images were taken at room temperature with an epifluorescent microscope (Nikon Eclipse TE).

Results

[0264] In order to overcome the above-mentioned problems, the present inventors came up with a hypothesis that endogenous activation of key transcription factors such as PAX6, MITF and OTX2 using CRISPR-dCas9/SAM will be enough to differentiate pluripotent stem cells into mature RPE tissue based on the knowledge search in the literature (FIG. 2). For this, sgRNA sequences (FIG. 3 and Table 2) were designed upstream of the target genes and top performing guide sequence were evaluated based on the maximal fold change achieved. After the initial screening, the present disclosure concentrated on the top performing candidates of sgRNA lentiviruses and demonstrated higher gene expression level (FIG. 4). Next, each of the top performing sgRNA lentiviruses of three target genes were added and the inventors were able to show multiplex activation of those genes in a single cell (FIG. 5). It was further showed that multiplexed endogenous activation of three genes and subsequent culture of cells in RPEM media resulted in pigmented, cobbled shaped foci of RPE cells at day 40 with all the RPE marker genes progressively upregulated over time. Importantly, the purity of the RPE population based on the PMEL17 expression is more than 96%. By this proposed method, a simple, robust and cost-effective protocol for RPE generation from pluripotent stem cells was achieved.

[0265] The RPE specific markers expression between day 21 hRPE (Lonza) and day 28 p1 of hiPSC-CRISPR/dCas9-SAM activated RPE cells were compared. The results demonstrated that iPSC-CRISPR activated RPE cells on day 28 gene expression patterns are similar to that of day 21 commercial human primary RPE cells (hRPE, Lonza) grown on laminin-coated 12-well plate (FIG. 7a). Specifically, the early eye gene markers Pax6, Mitf, Otx2, Lhx2 and Rax expression were similar to the hRPE cells. However, expression of pigmentation genes (Tyr and TyrP1) and mature markers (CRALBP, BEST1 and PEDF) expression were markedly higher in hRPE cells compared to the CRISPR activated RPE cells (FIG. 7a).

[0266] In order to improve the efficiency of RPE differentiation, the differentiation and expansion of iPSC-CRISPR activated RPE cells were tested on Laminin-521 (Ln521) coated 12-well plates. The efficiency of RPE marker expression of iPSC-CRISPR activated RPE cells were compared in both geltrax and Ln521 coated plates. The results show that Ln521 efficiently supports RPE differentiation (FIG. 7b) with robust expression of early eye-field genes (Pax6, Mitf, Otx2, Lhx2 and Rax), pigmentation genes (pmell7 & Tyrp2) and mature RPE markers (PEDF and BEST1). Based on this data, Ln521 coating was used for further studies.

[0267] Based on these studies (FIGS. 6 and 7), it is quite evident that activating the transcription factors (Pax6, Mitf and Otx2) in hiPSC cells can commit the cells towards RPE lineage. However, the uniformity and efficiency of RPE generation is hindered because of the addition of three individual sgRNAs in a cocktail possibly because different cells might receive the combination of different sgRNA dosage and fraction.

[0268] In order to overcome this, all three sgRNAs were incorporated in a single lentiviral vector as shown in FIG. 8a. This would allow each cell to activate all three transcription factors in unison. To identify the optimal concentration of triple sgRNA lentiviral infection, the iPSC-CRISPR dCas9 SAM cells were transduced with the triple sgRNA lentivirus at different concentrations. It was found that transduction using 9 μL of concentrated triple sgRNA virus yielded optimal expression of all three transcription factors (FIG. 8b). Next, to validate the hypothesis that triple sgRNA lentivirus has better RPE induction efficiency compared to the mix of individual lentiviruses (P+M+O), the experiment as shown in schematic FIG. 9a was carried out. It was observed that the expression of most of the RPE markers were slower with triple sgRNA transduced cells compared to P+M+O transduced cells on days 4, 10 and 18 (FIG. 9b). After replating the triple sgRNA cells on day 18, for the subsequent days, an increased expression of the activated transcription factors Pax6 and Mitf was observed, but the otx2 levels reduced as reported earlier (Ref. 11) (FIG. 10a). Pluripotency gene (Oct4-) decreased rapidly while the pigmentation genes (pMEL17, Tyr, TyrP1 and TyrP2) and mature markers (CRALBP, BEST1, RPE65 and PEDF) kept increasing throughout the period (FIG. 10a). Further examination of the cells in flow cytometry reveals similar phenomenon of high expression levels of pMEL17, Pax6 and Mitf, while the Otx2 protein expression levels were expressed at relatively consistent levels throughout the differentiation (FIG. 10b). More importantly, the morphology of pigmented clusters of cells in the 12-well plate was uniformly distributed throughout the well and the RPE signature cobblestone morphology was also present (FIG. 10c). These results confirms that the hypothesis of triple sgRNA design markedly improved RPE differentiation efficiency.

[0269] Next, in order to further optimize the protocol, different serum-free conditions were tested for the RPE maintenance media. The schematic of the differentiation protocol is shown in FIG. 11a. For this, the same protocol was maintained until day 18 and it was observed that the iPSC-CRISPR dCas9 SAM cells progressed gradually into RPE progenitor cells as observed with the increased expression of RPE-specific genes as observed earlier (FIG. 11b). After replating the RPE progenitor cells on day 18, the cells were maintained under two different serum-free formulations such as RPE maintenance media with, No FBS (0% FBS) or 5% KOSR. As a control, 5% FBS containing RPE was used as maintenance media. From this study, it is observed that 5% KOSR showed consistently higher RPE signature gene expression and the cells progressively matured into colonies comprising of a monolayer of pigmented polygonal cells and these pigmented cells within these colonies formed a cobble stone like sheets (FIGS. 11c and d). Immunostaining of cells enriched at day 21 of passage 1 iPSC-CRISPR dCas9 SAM derived RPE cells with mature tight junction markers ZO-1, N-Occludin, N-Cadherin and Bestrophin 1 showed that the cells were connected by tight junctions, properties which are highly characteristic of native RPE cells in vivo (FIG. 11e). Further immunostaining and flow cytometry analysis of activated transcription factors (Pax6, Mitf, Otx2) and pigmentation gene (pMEL17) showed higher and uniform expression, while pluripotency marker proteins (Oct4- and Tra-1-60) expression were low (FIGS. 11e and f). The optimized protocol disclosed herein was validated with hESC-CRISPR dCas9 SAM cells using triple sgRNA lentivirus and RPEM with 5% KOSR after replating the cells on day 18 and similar results was observed and RPE characteristics were as seen with iPSC CRIPSR dCas9-SAM cells (FIG. 12). This shows the robustness and reproducibility of this protocol with different pluripotent cells.

[0270] To further validate that activation of at least one (or all three) important to induce RPE generation, the RPE induction efficiency of triple sgRNA was compared with Mitf sgRNA only. Non-transduced cells was used as a control (i.e. No virus) as shown in FIG. 13a. It is evident from the data that, only triple sgRNA transduced cells generated RPE cells with characteristic RPE signature gene expression (FIG. 13b), pigmented cell clusters (FIG. 13c), typical cobble stone morphology (FIG. 13d) and protein expression (FIG. 13e). Overall, the present disclosure demonstrated that RPE cells can be rapidly and efficiently generated by activating only three transcription factors, Pax6, Mitf and Otx2 in pluripotent cells without the need for costly growth factors and small molecules.

[0271] Here, as a proof-of-concept the present study has demonstrated the activation of growth factors and cytokines (EPO, SCF, TPO, GM-CSF and G-CSF) using iPSC-CRISPR dCas9 SAM cells. For this, four different sgRNAs were designed for each of the growth factors/cytokines and constructed lentiviral vectors as mentioned previously (Table 4). For testing the activity of EPO expression, the lentiviruses of four different sgRNAs were produced and screened for the best performing guide in activating EPO expression. The iPSC-CRISPR dCas9 SAM cells were transduced with the unconcentrated lentivirus supernatant of the four sgRNAs individually and tested for their EPO gene expression using qPCR analysis on day 4 cells after transduction. It was found that g2 gave higher EPO gene expression as compared to the non-transduced control cells (FIG. 14a). Further, the spent media of the cells were collected from two wells of a 12-well plate on days 3 and 4 transduced with EPO_g2 and was stored in −20° C. Next, an Amicon Ultra-15 centrifugal filter was used with 10KDa cut-off membrane (EPO molecular weight: 21 KDa) to concentrate the EPO protein and the retentate washed with 1× PBS was used for ELISA assay. The collected data showed that EPO secreted from the iPSC-CRISPR dCas9 SAM cells transduced with EPO_g2 was detected by the commercial ELISA kit and the concentration was in the range of 47-51 IU/mL of EPO.

[0272] Similarly, the best performing sgRNA sequences for activating SCF, TPO, GM-CSF and G-CSF in iPSC-CRISPR dCas9 cells were screened (FIG. 15). The collected data showed, relatively lower expression of TPO, GM-CSF and G-CSF compared to SCF or EPO.

[0273] In the present study, EPO_g2 and SCF_g4 lentiviruses were concentrated according to previously described method. Transduction and selection of HEK-CRISPR dCas9 SAM cells were also shown. In the present disclosure, as shown in FIG. 16, the inventors have also stably transduced the EPO_g2 and SCF_g4 lentiviruses in HEK-CRISPR dCas9 SAM cells.

REFERENCES

[0274] 1. Li, L.; Hu, S.; Chen, X., Non-viral delivery systems for CRISPR/Cas9-based genome editing: Challenges and opportunities. Biomaterials 2018, 171, 207-218.

[0275] 2. Glass, Z.; Lee, M.; Li, Y.; Xu, Q., Engineering the Delivery System for CRISPR-Based Genome Editing. Trends Biotechnol 2018, 36 (2), 173-185.

[0276] 3. Konermann, S.; Brigham, M. D.; Trevino, A. E.; Joung, J.; Abudayyeh, O. O.; Barcena, C.; Hsu, P. D.; Habib, N.; Gootenberg, J. S.; Nishimasu, H.; Nureki, O.; Zhang, F., Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 2015, 517 (7536), 583-588.

[0277] 4. Yu, P.; Pan, G.; Yu, J.; Thomson, J. A., FGF2 sustains NANOG and switches the outcome of BMP4-induced human embryonic stem cell differentiation. Cell Stem Cell 2011, 8 (3), 326-34.

[0278] 5. Meyer, J. S.; Shearer, R. L.; Capowski, E. E.; Wright, L. S.; Wallace, K. A.; McMillan, E. L.; Zhang, S. C.; Gamm, D. M., Modeling early retinal development with human embryonic and induced pluripotent stem cells. Proc Natl Acad Sci U S A 2009, 106 (39), 16698-703.

[0279] 6. Radeke, M. J.; Peterson, K. E.; Johnson, L. V.; Anderson, D. H., Disease susceptibility of the human macula: differential gene transcription in the retinal pigmented epithelium/choroid. Exp Eye Res 2007, 85 (3), 366-80.

[0280] 7. Amram, B.; Cohen-Tayar, Y.; David, A.; Ashery-Padan, R., The retinal pigmented epithelium—from basic developmental biology research to translational approaches. Int J Dev Biol 2017, 61 (3-4-5), 225-234.

[0281] 8. Luo, M.; Chen, Y., Application of stem cell-derived retinal pigmented epithelium in retinal degenerative diseases: present and future. Int J Ophthalmol 2018, 11 (1), 150-159.

[0282] 9. da Cruz, L.; Fynes, K.; Georgiadis, 0.; Kerby, J.; Luo, Y. H.; Ahmado, A.; Vernon, A.; Daniels, J. T.; Nommiste, B.; Hasan, S. M.; Gooljar, S. B.; Carr, A. F.; Vugler, A.; Ramsden, C. M.; Bictash, M.; Fenster, M.; Steer, J.; Harbinson, T.; Wilbrey, A.; Tufail, A.; Feng, G.; Whitlock, M.; Robson, A. G.; Holder, G. E.; Sagoo, M. S.; Loudon, P. T.; Whiting, P.; Coffey, P. J., Phase 1 clinical study of an embryonic stem cell-derived retinal pigment epithelium patch in age-related macular degeneration. Nat Biotechnol 2018, 36 (4), 328-337.

[0283] 10. Buchholz, D. E.; Hikita, S. T.; Rowland, T. J.; Friedrich, A. M.; Hinman, C. R.; Johnson, L. V.; Clegg, D. O., Derivation of functional retinal pigmented epithelium from induced pluripotent stem cells. Stem Cells 2009, 27 (10), 2427-34.

[0284] 11. Buchholz, D. E.; Pennington, B. O.; Croze, R. H.; Hinman, C. R.; Coffey, P. J.; Clegg, D. O., Rapid and efficient directed differentiation of human pluripotent stem cells into retinal pigmented epithelium. Stem Cells Transl Med 2013, 2 (5), 384-93.

[0285] 12. Idelson, M.; Alper, R.; Obolensky, A.; Ben-Shushan, E.; Hemo, I.; Yachimovich-Cohen, N.; Khaner, H.; Smith, Y.; Wiser, O.; Gropp, M.; Cohen, M. A.; Even-Ram, S.; Berman-Zaken, Y.; Matzrafi, L.; Rechavi, G.; Banin, E.; Reubinoff, B., Directed differentiation of human embryonic stem cells into functional retinal pigment epithelium cells. Cell Stem Cell 2009, 5 (4), 396-408.

[0286] 13. Maruotti, J.; Sripathi, S. R.; Bharti, K.; Fuller, J.; Wahlin, K. J.; Ranganathan, V.; Sluch, V. M.; Berlinicke, C. A.; Davis, J.; Kim, C.; Zhao, L.; Wan, J.; Qian, J.; Corneo, B.; Temple, S.; Dubey, R.; Olenyuk, B. Z.; Bhutto, I.; Lutty, G. A.; Zack, D. J., Small-molecule-directed, efficient generation of retinal pigment epithelium from human pluripotent stem cells. Proceedings of the National Academy of Sciences 2015, 112 (35), 10950.

[0287] 14. Osakada, F.; Jin, Z. B.; Hirami, Y.; Ikeda, H.; Danjyo, T.; Watanabe, K.; Sasai, Y.; Takahashi, M., In vitro differentiation of retinal cells from human pluripotent stem cells by small-molecule induction. J Cell Sci 2009, 122 (Pt 17), 3169-79.

[0288] 15. Zahabi, A.; Shahbazi, E.; Ahmadieh, H.; Hassani, S. N.; Totonchi, M.; Taei, A.; Masoudi, N.; Ebrahimi, M.; Aghdami, N.; Seifinejad, A.; Mehrnejad, F.; Daftarian, N.; Salekdeh, G. H.; Baharvand, H., A new efficient protocol for directed differentiation of retinal pigmented epithelial cells from normal and retinal disease induced pluripotent stem cells. Stem Cells Dev 2012, 21 (12), 2262-72.

[0289] 16. Zhu, Y.; Carido, M.; Meinhardt, A.; Kurth, T.; Karl, M. O.; Ader, M.; Tanaka, E. M., Three-Dimensional Neuroepithelial Culture from Human Embryonic Stem Cells and Its Use for Quantitative Conversion to Retinal Pigment Epithelium. PLOS ONE 2013, 8 (1), e54552.

[0290] 17. Choudhary, P.; Booth, H.; Gutteridge, A.; Surmacz, B.; Louca, I.; Steer, J.; Kerby, J.; Whiting, P. J., Directing Differentiation of Pluripotent Stem Cells Toward Retinal Pigment Epithelium Lineage. Stem Cells Transl Med 2017, 6 (2), 490-501.

[0291] 18. Geng, Z.; Walsh, P. J.; Truong, V.; Hill, C.; Ebeling, M.; Kapphahn, R. J.; Montezuma, S. R.; Yuan, C.; Roehrich, H.; Ferrington, D. A.; Dutton, J. R., Generation of retinal pigmented epithelium from iPSCs derived from the conjunctiva of donors with and without age related macular degeneration. PLOS ONE 2017, 12 (3), e0173575.

[0292] 19. Zhang, K.; Liu, G.-H.; Yi, F.; Montserrat, N.; Hishida, T.; Esteban, C. R.; lzpisua Belmonte, J. C., Direct conversion of human fibroblasts into retinal pigment epithelium-like cells by defined factors. Protein & cell 2014, 5 (1), 48-58.

[0293] 20. D′3 ssio, A. C.; Fan, Z. P.; Wert, K. J.; Baranov, P.; Cohen, M. A.; Saini, J. S.; Cohick, E.; Charniga, C.; Dadon, D.; Hannett, N. M.; Young, M. J.; Temple, S.; Jaenisch, R.; Lee, T. I.; Young, R. A., A Systematic Approach to Identify Candidate Transcription Factors that Control Cell Identity. Stem Cell Reports 2015, 5 (5), 763-775.

Applications

[0294] Embodiments of the methods disclosed herein provide a fast, efficient and cheap way of programming a cell. Embodiments of the disclosed methods also seek to overcome the problems relating to methods of altering a differentiation status of a cell (by expressing genes and/or proteins in the cell).

[0295] As discussed in the background section, convention methods of expressing proteins are rife with problems. As an alternative, the present inventors found a simple but surprisingly effective and easy methods that are discussed in further detail in the present disclosure. For example, in various embodiments, the methods as describe herein may use suspension of human cells (such as human embryonic kidney (HEK) cells) instead of traditional host cells (such as CHO or bacterial cells) to overcome one or more of the limitations known in the art. The methods as described herein also advantageously capable of producing stable producer lines and very cost effective as the cost of media for culturing human cells (such as HEK cells) are lower than the cost of media for culturing traditional host cells (such as CHO or bacterial cells).

[0296] Furthermore, the use of CRISPR activation method to activate the genes to produce proteins endogenously, instead of recombinant DNA, also overcame many of the limitations known in the art.

[0297] Advantageously, the present disclosure demonstrates a simple method of differentiating a stem cell to a mature/differentiated cell (such as retinal pigment epithelial cells). In particular, the method advantageously only uses minimal set of transcription factors. For example, when the method differentiates human pluripotent stem cells to retinal pigment epithelium cells using CRISPR/dCas9-SAM mediated activation, minimal set of transcription factors is required.

[0298] Even more advantageously, the present disclosure demonstrates a method of altering the differentiation status of a cell without the use of growth factors and/or small molecules. That is, the present method is free of the use of growth factors and/or small molecules (either in any of the steps or in the solution/media used). This feature reduces the total costs of running the method and, thus, is a cost-effective method. For example, in one of the embodiment of the present disclosure, activation of one or more (such as three) key transcription factors (such as PAX6, MITF, and OTX2) is sufficient to generate retinal pigment epithelial cells without the need for costly growth factors or small molecules. The protocols are also free of laborious differentiation steps.

[0299] For one of the embodiments of the present disclosure, the inventors have generated unique sgRNA sequences that can specifically activate PAX6, MITF and OTX2 genes with higher fold change respectively. When one or more of these transcription factors are used to generate retinal pigment epithelial cells, the method advantageously generates desired cell in a short time period. This is illustrated in the appearance of cobblestone morphology of highly pure RPE cell cultures (>96% PMEL17) within 40 days of activation of transcription factors (TFs).

[0300] It will be appreciated by a person skilled in the art that other variations and/or modifications may be made to the embodiments disclosed herein without departing from the spirit or scope of the disclosure as broadly described. For example, in the description herein, features of different exemplary embodiments may be mixed, combined, interchanged, incorporated, adopted, modified, included etc. or the like across different exemplary embodiments. The present embodiments are, therefore, to be considered in all respects to be illustrative and not restrictive.