Generation of induced pluripotent cells by crispr activation

Abstract

To provide methods and compositions for reprogramming adult somatic cells into induced pluripotent stem cells by targeting and remodeling endogenous gene loci without relying on ectopic expression of transcription factors.SOLUTION: A method of generating an induced pluripotent stem cell (iPSC) comprises: (a) targeting at least one endogenous gene locus using at least one single guide RNA (sgRNA) in a non-iPSC; and (b) remodeling the endogenous gene locus in the non-iPSC using a CRISPR activation system and the at least one sgRNA; thereby generating an iPSC.SELECTED DRAWING: Figure 1-1

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 62 / 611,202, filed December 28, 2017, the disclosure of which is incorporated herein by reference in its entirety.

[0002] The ability to reprogram adult somatic cells into pluripotent stem cells through the regulation of specific transcription factors is of great importance for basic biological research and holds great promise for regenerative medicine, where induced pluripotent stem cells (iPSCs) can be differentiated into any cell type in the body to treat numerous diseases and disorders. A continuing challenge in this field has been to generate iPSCs without relying on ectopic expression of transcription factors. The present disclosure provides novel methods and compositions for inducing pluripotent stem cells by targeting and reconstituting endogenous loci. [Background technology]

[0003] Pluripotent stem cells hold considerable promise for regenerative medicine. It is of great importance to better understand how remodeling of endogenous chromatin leads to pluripotency induction. Conventionally, differentiated somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by ectopic expression of Oct4, Sox2, Klf4, and c-Myc (OSKM) (Takahashi and Yamanaka, 2006). Overexpression of Oct4, Sox2, and Klf4 first binds and globally remodels endogenous loci throughout the genome (Soufi et al., 2012), ultimately leading to the establishment of pluripotency regulatory circuits.

[0004] However, it remains largely unknown which exact remodeling events to endogenous chromatin trigger reprogramming towards pluripotency. For one, it is not clear whether simultaneous remodeling of multiple pluripotency-associated loci is required or whether the very remodeling of a single locus is sufficient for iPSC induction. In addition, Oct4, Sox2, and Klf4 target distal elements of many genes required for reprogramming (Soufi et al., 2012), but it is poorly understood how remodeling of these distal elements affects pluripotency induction. Furthermore, although epigenetic remodeling is a central mechanism of cellular reprogramming (Smith et al., 2016), it remains to be determined whether iPSC induction can be initiated by epigenetic manipulation of any defined endogenous locus. Finally, due to methodological limitations, there is no direct evidence that pluripotency can be induced by the very remodeling of an endogenous locus.

[0005] Recently, the bacterial-derived type II clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated 9 (Cas9) nuclease system (CRISPR / Cas9 system) has been repurposed as a powerful tool for gene editing in mammalian cells (Cong et al., 2013; Jinek et al., 2012; Mail et al., 2013b). A nuclease-inactivated form of Cas9 (dCas9) has been engineered as a programmable synthetic transcription factor when fused with a transactivation domain. This system has been termed the CRISPR-activated (CRISPRa) system (Chavez et al., 2015; Gilbert et al., 2013; Konermann et al., 2015; Tanenbaum et al., 2014; Zalatan et al., 2015). This system has been reported to be able to target silenced chromosomal loci with high precision and act as pioneering factors to promote downstream gene transcription (Polstein et al., 2015). These features make the CRISPRa system an advantageous tool for precisely reconstructing endogenous chromatin loci for cell reprogramming from one lineage to another lineage-specific cell (Black et al., 2016; Chakraborty et al., 2014). However, it remains unclear whether and / or how this system can function to generate induced pluripotent stem cells with a full range of differentiation potential for cells of all lineages. Summary of the Invention

[0006] The present disclosure is based on the present invention's discovery that induced pluripotent stem cells (iPSCs) can be generated by targeting and reconstructing selected endogenous loci of somatic cells, and the present disclosure is directed to a method of generating iPSCs, comprising, in part, using at least one single guide RNA (sgRNA) in non-iPSCs to target at least one endogenous locus, and using a CRISPR activation system and at least one sgRNA to reconstruct selected endogenous loci in non-iPSCs to generate iPSCs. In some embodiments, the above method of generating iPSCs further comprises contacting the non-iPSC cells undergoing reprogramming with small molecules comprising TGFβR inhibitors, GSK3 inhibitors, MEK inhibitors and ROCK inhibitors, and optionally further comprises contacting the generated iPSCs with small molecules comprising GSK3 inhibitors, MEK inhibitors and ROCK inhibitors but not including TGFβR inhibitors.

[0007] In some embodiments, the CRISPR activation system comprises a nuclease-inactivated Cas9 (dCas9) fused with at least one transcription activator. In other embodiments, the CRISPR activation system further comprises a tandem array of peptides that link dCas9 to at least one transcription activator. In one embodiment, the tandem array of peptides is a SunTag array. In one embodiment, the at least one transcription activator is a tetramer of herpes simplex VP16 transcription activator domain (VP64). In one embodiment, the CRISPR activation system is dCas9-SunTag-VP64.

[0008] In other aspects, the disclosure provides a CRISPR activation system comprising dCas9, a SunTag array fused to dCas9, and at least one acetyltransferase activity domain of p300 (p300 core) associated with the SunTag array.

[0009] In other aspects, the present disclosure provides methods for generating iPSCs using a CRISPR activation system including dCas9, a SunTag array, and a p300 core.

[0010] In some embodiments, the at least one endogenous locus is Oct4, Sox2, Klf4, c-Myc, Lin28, Nanog, Nr5a2, Glis1, Cebpa, or any combination thereof.

[0011] In some embodiments, a single endogenous locus is targeted and reconstituted. In some embodiments, the single endogenous locus is Oct4 or Sox2.

[0012] In some embodiments, at least one sgRNA is an Oct4 promoter targeting sgRNA, an Oct4 enhancer targeting sgRNA, a Sox2 promoter targeting sgRNA, or a combination thereof. In some embodiments, the Oct4 promoter targeting sgRNA is selected from SEQ ID NOs: 1-6, 57, and 58. In some embodiments, the Oct4 enhancer targeting sgRNA is selected from SEQ ID NOs: 7-11. In some embodiments, the Sox2 gene targeting sgRNA is selected from SEQ ID NOs: 12-21 and 59.

[0013] In one embodiment, at least one sgRNA is a Klf4 gene targeted sgRNA. In some embodiments, the Klf4 gene targeted sgRNA is selected from SEQ ID NOs: 22-31 and 60. In one embodiment, at least one sgRNA is a c-Myc gene targeted sgRNA. In some embodiments, the c-Myc gene targeted sgRNA is selected from SEQ ID NOs: 32-41 and 61. In one embodiment, at least one sgRNA is a Nr5a2 gene targeted sgRNA. In some embodiments, the Nr5a2 gene targeted sgRNA is selected from SEQ ID NOs: 42-45. In one embodiment, at least one sgRNA is a Glis1 gene targeted sgRNA. In some embodiments, the Glis1 gene targeted sgRNA is selected from SEQ ID NOs: 46-50. In one embodiment, at least one sgRNA is a Cebpa gene targeted sgRNA. In some embodiments, the sgRNA targets the Cebpa gene selected from SEQ ID NOs: 51-56. In some embodiments, the sgRNA targets the Lin28 promoter. In some embodiments, the sgRNA targets the Lin28 promoter comprises SEQ ID NO: 62. In some embodiments, the sgRNA targets the Nanog promoter. In some embodiments, the sgRNA targets the Nanog promoter comprises SEQ ID NO: 63. In some embodiments, the sgRNA targets the EEA motif. In some embodiments, the sgRNA targets the EEA motif comprises SEQ ID NO: 64.

[0014] In some embodiments, the non-iPSC is a somatic cell, such as a fibroblast, a skin cell, an umbilical cord blood cell, a peripheral blood cell, or a renal epithelial cell.In some embodiments, the non-iPSC is a mammalian cell.In other embodiments, the non-iPSC is a human cell. [Brief description of the drawings]

[0015] [Figure 1-1]Figure 1 shows the establishment of pluripotency networks in mouse embryonic fibroblasts (MEFs) by gene activation. (A) Scheme showing dCas9-SunTag-VP64 complex in gene activation. (B) Scheme showing the reprogramming procedure of OG2 MEFs. (C) OG2 MEFs were reprogrammed to form EGFP-positive colonies. MEF morphology at day 0 and reprogrammed colonies at days 7 and 15 are shown (scale bar, 200 μm). (D) Endogenous Oct4 and Sox2 transcription over 12 days. Data represent mean ± SD (n=4). p values ​​were determined by one-way ANOVA with Dunnett's test. **p<0.01. (E) Colonies showing EGFP signal in situ and at passages 1 and 20 (scale bar, 200 μm). (F) Nanog, Sox2, and SSEA-1 staining in EGFP-positive colonies (scale bar, 200 μm). (G) Pluripotency gene expression in established CRISPR iPSCs and R1 mouse embryonic stem (R1 ES) cells. [Figure 1-2] Same as above. [Figure 2-1]We show that activation of the Sox2 gene is sufficient to generate iPSCs in MEFs. (A) Scheme showing sgRNA targeting sites in the Sox2 promoter along with binding peaks of transcription factors (Oct4, Sox2, Nanog), histone acetyltransferase p300, Mediator complex, and distribution of histone H3K27ac (Whyte et al., 2013). (B) Detection of Sox2 protein by immunofluorescence staining at day 4 (scale bar: 100 μm). (C) Activation of Sox2 in the presence of the indicated sgRNAs. O-71 and O-127 target the Oct4 promoter, while S-84, S-136, and S-148 target the Sox2 promoter. Data represent the mean ± SD (n = 4). p values ​​were determined by unpaired t-test. **p < 0.01. (D) Number of colonies generated using sgRNA O-71, O-127, S-84, S-136, or S-148 to target Oct4 or Sox2 promoters. Three independent experiments are shown. (E) Generation of EGFP-positive colonies and iPSC lines in situ by activating the Sox2 gene with S-84 (scale bar, 200 μm). (F) Nanog, Sox2, and SSEA-1 staining in EGFP-positive colonies generated with S-84 (scale bar, 200 μm). (G) Comparison of pluripotency gene expression in S-17 cell lines and R1 ES cells. (H) Male karyotype of S-17 lines. (I)-(K) In vivo characterization of pluripotent S-17 lines. Chimeric mice were generated using S-17 cells (J), and these cells contributed to gonadal tissue represented by a strong EGFP signal (I) and were competent for germline transmission (K). [Figure 2-2] Same as above. [Figure 3-1]We demonstrate that reconstitution of the Sox2 promoter triggers reprogramming of S-17 MEFs towards pluripotency. (A) Scheme showing the generation of S-17 MEFs. (B) Sox2 activation in S-17 MEFs with or without doxycycline over 12 days. (C) Histone H3K27 acetylation levels at the Sox2 promoter at days 0, 4, 8, and 12. (D) Detection of Sox2 expression by immunofluorescence staining in the presence of doxycycline (scale bar: 100 μm). (E) S-17 MEFs were reprogrammed to form EGFP-positive colonies and iPSC lines were established (scale bar: 200 μm). (F) Comparison of the efficiency of lentiviral SunTag reprogramming and S-17 MEF reprogramming. (G) Gene expression of Oct4, Nanog, and Rex1 over 12 days in S-17 MEF reprogramming. (H) Histone H3K27 acetylation levels at Oct4, Nanog, and Rex1 promoters at days 0, 4, 8, and 12. (I) Histone H3K27 acetylation levels at Oct4 enhancer at days 0, 4, 8, and 12. (J) Sox2 dependency in S-17 MEF reprogramming. Four different concentrations of doxycycline (0, 0.01, 0.1, and 1 μg / ml) were used to show Sox2 activation (left) and reprogramming efficiency (right). (K) Synergistic effect of Oct4 and Sox2 reconstitution in S-17 MEF reprogramming. Oct4 gene activation (left) and reprogramming efficiency (right) were examined. (L) Total (left) and endogenous Sox2 (right) expression at days 4 and 12 when pluripotency genes were overexpressed (OE). Three conditions were tested: Oct4 only, Sox2 only, and OSK (Oct4, Sox2, and Klf4), and overexpression of mCherry (mCh) was used as a control. (M) Comparison of reprogramming efficiency in S-17 MEFs with overexpression of pluripotency genes. Data in (B), (C), (G), (H), (I), and (K) represent the mean ± SD (n=4).The p-value for (B) was determined by two-way ANOVA with Bonferroni test, the p-values ​​for (C), (G), (H), and (I) were determined by one-way ANOVA with Dunnett's test, and the p-value (K) was determined by unpaired t-test. **p<0.01; *p<0.05. [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Figure 4-1]We demonstrate simultaneous reconstitution of Oct4 promoter and enhancer to reprogram MEFs to iPSCs. (A) Scheme showing sgRNA targeting sites in Oct4 promoter and enhancer along with binding peaks of transcription factors (Oct4, Sox2, Nanog), histone acetyltransferase p300, Mediator complex, and distribution of histone H3K27ac and DNase hypersensitive sites (DHS) (Whyte et al., 2013). (B) Histone H3K27 acetylation levels in Oct4 enhancer and promoter at day 4. Three different sites were examined: 2.7 kb, 1.4 kb, and 0.2 kb upstream of the transcription start site. (C) Endogenous Oct4 transcription in the presence of O-127, O-2066, or O-127-2066 sgRNA over 16 days. (D) Number of colonies generated from reconstitution of Oct4 promoter (O-127), enhancer (O-2135, O-2066, O-1965), or promoter and enhancer simultaneously (O-127-2135, O-127-2066, O-127-1965). Four independent experiments are shown. No colonies were observed in O-127-2135 cultures in experiment 3 and O-127-2066 / 1965 in experiment 4. (E) Morphology of EGFP-positive colonies in situ and in P0 iPSCs from reconstitution of Oct4 promoter and enhancer simultaneously (O-127-2066) (scale bar, 200 μm). (F) Expression of Nanog, Sox2, and Rex1 in Oct4-EGFP-positive colonies (scale bar, 200 μm). (G) Comparison of pluripotency gene expression in D-9 cell line and R1 ES cells. (H) Karyotype analysis of D-9 line. (I)-(K) In vivo characterization of pluripotent D-9 line. Chimeric mice were generated using D-9 cells (J), and these cells contributed to gonadal tissue represented by strong EGFP signal (I) and were suitable for germline transmission (K). Data in (B) and (C) represent mean ± SD (n=4). p values ​​were determined by unpaired t-test. **p<0.01. [Figure 4-2] Same as above. [Figure 5-1]Gene activation by the SunTag system in differentiating mouse ES cells and MEFs. (A), (B) Scheme showing sgRNA targeting sites in the Oct4 promoter and enhancer (A) and the Sox2 promoter (B) along with binding peaks of transcription factors (Oct4, Sox2, Nanog), histone acetyltransferase p300, Mediator complex, and distribution of histone H3K27ac (Whyte et al., 2013). (C) Genomic DNA sequence 500 bp upstream and 100 bp downstream of the Sox2 transcription start site (SEQ ID NO: 175). Binding sites of sgRNA (blue) and transcription factors (shading) are highlighted. (D), (E) Transcription activation of Oct4, Sox2, Klf4, c-Myc, Nr5a2, Glis1, and Cebpa using sgRNAs in differentiating mouse ES cells. Schematic diagram showing the experimental procedure (D) and fold transcription activation using each sgRNA (E) are shown. Gal4 sgRNA was used as a negative control. (F), (G) Transcriptional activation of Oct4 and Sox2 using sgRNAs in MEFs. Experimental procedure (F) and fold transcriptional activation using selected sgRNAs (G) are shown. Gal4 sgRNA was used as a negative control. sgRNAs targeting Oct4 promoter (Oct4 Pro) include O-71 and O-127, and sgRNAs targeting Oct4 enhancer (Oct4 Enh) include O-1965, O-2066, and O-2135. sgRNAs targeting Sox2 promoter (Sox2 Pro) include S-84, S-136, and S-148. (H) Activation of Oct4 and Sox2 using a small molecule combination of Parnate, Chir99021, A83-01, and forskolin (PCAF). Data in (E), (G), and (H) represent the mean ± SD (n = 4). p values ​​for (E) and (G) were determined by one-way analysis of variance with Dunnett's test, and p value for (H) was determined by unpaired t test. *p < 0.05; **p < 0.01; ns, not significant. [Figure 5-2] Same as above. [Figure 5-3] Same as above. [Figure 6-1]We show that activation of the Sox2 gene is sufficient to generate iPSCs in MEFs. (A) Number of EGFP-positive colonies when sgRNAs targeting the indicated genes were taken out of the sgRNA pool. (B) Comparison of activation using one sgRNA and multiple sgRNAs of Oct4, Sox2, and Glis1 on day 3. (C) Transcriptional activation of Oct4, Sox2, and Glis1 using O-127, S-84, G-215, or all (OSG) on day 3. (D) Morphology of EGFP-positive colonies in situ and P0 iPSCs targeting Oct4, Sox2, and Glis1 promoters together (scale bar, 200 μm). (E) Nanog, Sox2, and SSEA-1 staining of EGFP-positive colonies from targeting Oct4, Sox2, and Glis1 promoters together (scale bar: 200 μm). (F) Number of EGFP-positive colonies generated from targeting of Oct4, Sox2, and Glis1 promoters. Three independent experiments are shown. (G) Examination of off-target effects of S-84 sgRNA. Transcription of the top 10 predicted targets was examined. Data in (B) and (C) represent mean ± SD (n = 4). p values ​​were determined by unpaired t-test. **p<0.01; *p<0.05; ns, not significant. [Figure 6-2] Same as above. [Figure 7-1]We demonstrate that reconstitution of the Sox2 promoter triggers reprogramming of MEFs towards pluripotency. (A) Comparison of Sox2 activation in OG2 and 129 MEFs with S-84. Data represent the mean ± SD (n=4). p values ​​were determined by unpaired t-test. **p<0.01. (B) Staining of Sox2 protein in 129 MEFs at day 4 (scale bar: 200 μm). (C) Generation of reprogrammed colonies in 129 MEFs (scale bar: 200 μm). (D) Determination of copy number of sgRNA cassette in 12 established CRISPR iPSC lines. Clone 8 is S-17 line as indicated. (E) Flow cytometry examination of BFP in S-17 MEFs. (F) Percentage of Sox2 positive cells at day 4 with or without doxycycline for S-17 MEF reprogramming. (G) Examination of off-target effects of S-84 sgRNA in S-17 MEF reprogramming. Transcription of the top 10 predicted targets was examined by qPCR. (H) Coefficient of variation (CV) comparison of lentiviral SunTag and S-17 MEF reprogramming efficiency. (I) Comparison of reprogramming efficiency with and without PCAF cocktail. (J) Histone H3K27 acetylation levels at Sox2 promoter at day 4 when pluripotency genes were overexpressed (OE). Three conditions were tested: Oct4 only, Sox2 only, OSK (Oct4, Sox2, and Klf4), and mCherry overexpression (mCh OE) was used as a negative control. (K) Coefficient of variation (CV) comparison of reprogramming efficiency of OSK overexpression and S-17 MEF. (L) S-17 TTFs were reprogrammable. Morphology of S-17 TTFs at day 0 and reprogrammed colonies at days 7 and 14 are shown along with activation of Oct4-EGFP (scale bar: 200 μm). [Figure 7-2] Same as above. [Figure 8-1]Simultaneous reconstruction of Oct4 promoter and enhancer demonstrates reprogramming MEFs to iPSCs. (A) Functional validation of dual sgRNA cassette in differentiating ES cells by introducing two sgRNAs targeting Sox2 (S-148) and c-Myc (M-154). (B) Oct4 protein staining at day 8 of reprogramming (scale bar: 200 μm). (C) Examination of off-target effects of O-127 (left) and O-2066 (right). Transcription of the top 10 predicted targets of each sgRNA was examined by qPCR. (D) Transcription of total (left) and endogenous (right) Oct4 at days 4 and 12 when pluripotency genes were overexpressed (OE). Two conditions were tested: Oct4 alone and OSK (Oct4, Sox2, and Klf4). mCherry was used as a negative control. (E) Scheme representing the dCas9-SunTag-p300 core and its function. (F) Histone H3K27 acetylation levels at Oct4 enhancer and promoter at day 5 for dCas9-SunTag-VP64 and dCas9-SunTag-p300 core systems. Three different sites were examined: 2.7 kb, 1.4 kb, and 0.2 kb upstream of the transcription start site. (G) Transcriptional activation of Oct4 using dCas9-SunTag-VP64 or dCas9-SunTag-p300 core over 15 days. (H) Morphology of in situ EGFP-positive colonies and P1 iPSCs (D-16) generated by engineering acetylation of Oct4 promoter and enhancer with dCas9-SunTag-p300 core (scale bar, 200 μm). (I) Comparison of pluripotency gene expression in D-16 cell line and R1 ES cells. Data in (A), (D), and (G) represent mean ± SD (n=4). p values ​​for (A) and (D) were determined by two-way ANOVA with Bonferroni test, and p value for (G) was determined by unpaired t test. **p<0.01; ns, not significant. [Figure 8-2] Same as above. [Figure 9]We demonstrate that CRISPRa generated human iPSC colonies that adopt and maintain pluripotent morphology. The top panel shows that the morphological signature of hiPSCs is more robust when EEA is included in the iPSC generation compared to hiPSCs generated without EEA (bottom panel at day 28). [Figure 10] 10 demonstrates expression of pluripotency genes in CRISPRa-generated human iPSC colonies. A: Day 28 hiPSCs generated without EEA and with MOI=1.2. and B: Day 28 hiPSCs generated with EEA and MOI=1.2. C: Day 21 hiPSCs generated with EEA and MOI=6. [Figure 11] We demonstrate that the endogenous pluripotency program is activated over time in the reprogrammed population, as indicated by increased expression of both targeted and non-targeted endogenous genes, including Oct4(Pou5f1), Sox2, Lin28, Nanog and Rex1. [Figure 12] We demonstrate that expression of the pluripotency gene TRA-1-81 increases over time within the reprogrammed population as shown by flow cytometry. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] This disclosure relates to the generation of induced pluripotent stem cells (iPSCs) by targeting and reconstituting endogenous loci using a CRISPR activation system.

[0017] It is to be understood that the present disclosure is not limited to the particular embodiments described, as such may vary. The scope of the present disclosure will be limited only by the appended claims, and it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0018] The detailed description of the present disclosure is divided into various sections for the convenience of the reader only, and the disclosure in any section can be combined with that in another section.Although any method and material similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred method and material are described here.All publications mentioned in this specification are incorporated by reference to disclose and describe the method and / or material related to which the publication is cited.

[0019] I. Definition To facilitate understanding of this disclosure, several terms are defined below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms such as "a", "an", "the" and the like are not intended to refer to only a single entity, but include a general class in which a specific example can be used to describe.

[0020] As used herein, the term "generate" refers to the creation and / or alteration of a biological composition (e.g., a cell) from its naturally occurring state by any manipulation, including but not limited to altering chromosomal structure, the nucleotide sequence of a polynucleotide (e.g., DNA or RNA), the amino acid sequence of a polypeptide (e.g., a protein), the transcription level of a gene, the epigenetic modification of a chromosomal region, the transcription level of a protein, and the transcription level of an RNA.

[0021] As used herein, the term "reprogramming" or "dedifferentiation" or "increasing cell potential" or "increasing developmental potential" refers to a method of increasing the potential of a cell or dedifferentiating a cell into a less differentiated state. For example, a cell with increased cell potential has more developmental plasticity (i.e., can differentiate into more cell types) compared to the same cell in a non-reprogrammed state. In other words, a reprogrammed cell is a cell in a less differentiated state than the same cell in a non-reprogrammed state.

[0022] The term "pluripotent" or "pluripotency" refers to the ability of a cell to self-renew and differentiate into multiple tissues or all lineage cell types in the body. For example, embryonic stem cells are a type of pluripotent stem cell that can form cells from each of the three germ layers, ectoderm, mesoderm, and endoderm. Pluripotency is a continuum of developmental potential ranging from incomplete or partially pluripotent cells (e.g., epiblast stem cells or EpiSCs) that cannot give rise to a complete organism, to more primitive, more pluripotent cells that can give rise to a complete organism (e.g., embryonic stem cells).

[0023] Pluripotency can be determined, in part, by assessing the pluripotent properties of a cell, including, but not limited to, (i) pluripotent stem cell morphology, (ii) unlimited self-renewal potential, (iii) expression of pluripotent stem cell markers such as SSEA1 (mouse only), SSEA3 / 4, SSEA5, TRA1-60 / 81, TRA1-85, TRA2-54, GCTM-2, TG343, TG30, CD9, CD29, CD133 / prominin, CD140a, CD56, CD73, CD90, CD105, OCT4, NANOG, SOX2, CD30 and / or CD50, (iv) ability to differentiate into all three somatic lineages (ectoderm, mesoderm, endoderm), (v) formation of teratomas composed of the three somatic lineages, and (vi) formation of embryoid bodies composed of cells of the three somatic lineages.

[0024] Two types of pluripotency have been described so far: a "primed" or "metastable" state of pluripotency, similar to epiblast stem cells (EpiSCs) of late blastocysts, and a "naive" or "ground" state, similar to the inner cell mass of pluripotent early / preimplantation blastocysts. While both pluripotent states exhibit characteristics as described above, the naive or ground state additionally exhibits (i) pre-inactivation or reactivation of the X chromosome in female cells, (ii) enhanced clonality and survival during single cell culture, (iii) an overall reduction in DNA methylation, (iv) reduced H3K27me3 repressive chromatin mark deposition on developmental regulatory gene promoters, and (v) reduced expression of differentiation markers compared to primed pluripotent cells. Standard methodologies of cell reprogramming, in which exogenous pluripotency genes are introduced into somatic cells, expressed, and then either silenced or removed from the resulting pluripotent cells, generally appear to have the characteristics of a primed state of pluripotency. Under standard pluripotent cell culture conditions, such cells remain in a primed state and exhibit characteristics of the ground state unless exogenous transgene expression is maintained.

[0025] As used herein, "induced pluripotent stem cells" or iPSCs means that stem cells are generated from induced or altered differentiated adult, neonatal, or fetal cells, i.e., artificially reprogrammed into cells that are less differentiated, exhibit pluripotent characteristics, such as expression of specific stem cell genes and proteins, chromatin methylation patterns, embryoid body formation, teratoma formation, viable chimera formation, and can differentiate into all three germ layers, endoderm (e.g., stomach lining, digestive tract, lungs), mesoderm (e.g., muscle, bone, blood, urogenital), or ectoderm (e.g., epidermal tissue and nervous system), or tissues of the dermis layer. Generated iPSCs do not refer to naturally occurring cells.

[0026] As used herein, the term "pluripotent stem cell morphology" refers to the classical morphological characteristics of embryonic stem cells. Normal embryonic stem cell morphology is characterized by a round and small shape, with a high nucleus-to-cytoplasm ratio, prominent presence of nucleoli, and typical intercellular spacing.

[0027] As used herein, the term "non-iPSC" refers to any cell in a differentiated or partially differentiated state, which is not an embryonic stem cell (ESC) or an iPSC. Non-iPSCs may be derived from any non-germline tissue of the body, including internal organs, skin, bone, blood, nervous tissue, and connective tissue.

[0028] As used herein, "CRISPR / Cas system" refers to a bacterial derived system for defense against foreign nucleic acids. CRISPR / Cas systems are found in a wide range of eubacterial and archaeal organisms and include type I, II, and III subtypes. CRISPR-associated nucleases are derived from families including cas, cpf, cse, csy, csn, csd, cst, csh, csa, csm, and cmr. Wild-type type II CRISPR / Cas systems utilize RNA-mediated nucleases in complex with CRISPR RNA (crRNA) and sometimes transactivate crRNA (tracrRNA) to recognize and cleave foreign nucleic acids. For example, in the case of Cas9, when both crRNA and tracrRNA are required, the crRNA and tracrRNA can be incorporated into a single guide RNA (sgRNA) molecule that, when combined with Cas9, complementarily finds and cleaves DNA targets through base pairing between the guide sequence in the sgRNA and the target DNA sequence. As used herein, the term "nuclease-inactivated Cas9" or "dCas9" refers to a modified Cas9 nuclease whose nuclease activity has been abolished by mutating residues in its catalytic RuvC and HNH domains. Disabling the catalytic domain converts Cas9 from an RNA-programmable nuclease to an RNA-programmable DNA recognition complex that can deliver effectors or markers to specific target DNA sequences. Similar manipulations can be applied to other CRISPR-associated nucleases. As used herein, the term "CRISPR-activated system" or "CRISPRa" refers to a CRISPR / Cas system modified to upregulate gene transcription, where the inactivated Cas nuclease is fused to a transcription activator such as herpes simplex VP16 transcription activation domain, VP64 activator (an artificial tetramer of VP16), activation domain of p65, synergistic combinations of multiple transcription activators, scaffold peptides that recruit multiple copies of transcription activators, or histone acetyltransferases.

[0029] As used herein, the term "single guide polynucleotide" or "sgPNA" refers to a DNA, RNA, or mixed DNA / RNA sequence used in combination with a CRISPR / Cas system. The term "single guide RNA" or "sgRNA" refers to an RNA sequence used in combination with a CRISPR / Cas system. The sgPNA contains a binding site for a CRISPR-associated nuclease, including Cas9, and a guide sequence that is complementary to a desired target DNA sequence. Base pairing between the sgPNA and the target DNA sequence allows the CRISPR-associated nuclease to be recruited to the DNA sequence.

[0030] As used herein, the term "targeting" refers to the process of identifying a nucleic acid sequence (i.e., a target) of any composition and / or length in a chromosomal locus, including but not limited to a gene, promoter, enhancer, open reading frame, or other chromosomal region. Then, an sgRNA is designed and synthesized that includes a guide sequence that is complementary to the pre-identified nucleic acid sequence. In some embodiments, the sgRNA directs the CRISPR activation system to the vicinity of the endogenous locus that has the pre-identified nucleic acid sequence based on the complementary base pairing of the sgRNA to the pre-identified nucleic acid sequence.

[0031] As used herein, the term "remodel" or "remodeling" refers to any modification of the expression of a gene at the transcriptional level. Exemplary gene remodeling includes, but is not limited to, alteration of the chromatin structure of a gene, recruitment of transcription activators to the promoter of a gene, recruitment of transcription activators to the enhancer of a gene, and alteration of the histone modification of a gene by a specific enzyme (e.g., histone acetyltransferase).

[0032] II.Cells The present invention is based on the discovery that non-iPSCs, e.g. somatic cells, can be reprogrammed to pluripotency by activating pluripotency-associated genes, either alone or in combination, at their endogenous loci through CRISPR activation.

[0033] Non-iPSCs of the present disclosure include any cell in the body that is not a stem cell, a germ cell, or an iPSC. Non-limiting examples of non-iPSCs are somatic cells derived from non-germline tissues of the body, including viscera, skin, bone, blood, nervous tissue, and connective tissue. In some embodiments, the somatic cells are fetal somatic cells. In some embodiments, the somatic cells are adult somatic cells. In some embodiments, the non-iPSCs are derived from cell types that are easily accessible and require minimal invasion, such as fibroblasts, skin cells, umbilical cord blood cells, peripheral blood cells, and kidney epithelial cells.

[0034] The non-iPSCs of the present disclosure may be derived from a mammal, preferably a human, including but not limited to non-human primates, murines (i.e., mice and rats), dogs, cats, horses, cows, sheep, pigs, goats, etc. In some embodiments, the non-iPSCs are human non-iPSCs.

[0035] III. Targeting and reconstitution of endogenous loci The present disclosure relates to targeting and reconstructing endogenous loci using a CRISPR activation system with at least one sgRNA that targets a desired locus. In some embodiments, the endogenous locus is a pluripotency-related gene. In other embodiments, the endogenous locus is a non-pluripotency-related gene. Non-limiting examples of pluripotency-related genes are Oct4, Sox2, Nanog, Klf4, c-Myc, Lin28, Nr5a2, Glis1, Cebpa, Esrrb, Rex1. In some embodiments, the endogenous locus is Oct4 or Sox2. In some embodiments, the endogenous locus is a combination of Oct4, Sox2, Nanog, Klf4, c-Myc and Lin28.

[0036] In some embodiments, the method for generating iPSCs comprises using at least one sgRNA in conjunction with a CRISPR activation system that targets pluripotency-associated genes.In some embodiments, the sgRNA targets pluripotency-associated genes selected from the group consisting of Oct4, Sox2, Nanog, Klf4, c-Myc, Lin28, Nr5a2, Glis1, Cebpa, Esrrb, and Rex1.In some embodiments, the sgRNA targets pluripotency-associated genes selected from the group consisting of Oct4, Sox2, Nanog, Klf4, c-Myc, Lin28, Nr5a2, Glis1, and Cebpa.

[0037] In some embodiments, sgRNA targets the promoter region of pluripotency-associated genes.In some embodiments, sgRNA targets the enhancer region of pluripotency-associated genes.In some embodiments, sgRNA targets the promoter region of Oct4 gene, the enhancer region of Oct4 gene, the promoter region of Sox2 gene, or combinations thereof.

[0038] In some embodiments, the sgRNA targets a region of the Oct4 promoter and / or enhancer region about 1 to about 5000-bp upstream of the Oct4 transcription start site (TSS), about 100-bp to about 4000-bp upstream of the Oct4 TSS, about 200-bp to about 3000-bp upstream of the Oct4 TSS, about 400-bp to about 4000-bp upstream of the Oct4 TSS, about 500-bp to about 3000-bp upstream of the Oct4 TSS, about 500-bp to about 3000-bp upstream of the Oct4 TSS, about 600-bp to about 2000-bp upstream of the Oct4 TSS, or about 700-bp to about 1000-bp upstream of the Oct4 TSS. In some embodiments, the sgRNA is selected from the group consisting of about 10-bp, about 20-bp, about 30-bp, about 40-bp, about 50-bp, about 60-bp, about 70-bp, about 80-bp, about 90-bp, about 100-bp, about 110-bp, about 120-bp, about 130-bp, about 140-bp, about 150-bp, about 160-bp, about 170-bp, about 180-bp, about 190-bp, about 200-bp, about 210-bp, about 220-bp, about 230-bp, about 240-bp, about 250-bp, about 260-bp, about 270-bp, about 280-bp, about 290-bp, about 300-bp, about 310-bp, about 320-bp, about 330-bp, about 340-bp, about 350-bp, about 360-bp, about 370-bp, about 380-bp, about 390-bp, about 400-bp, about 410-bp, about 420-bp, about 430-bp, about 440-bp, about 450-bp, about 460-bp, about 470-bp, about 480-bp, about 490-bp, about 500-bp, about 510-bp, about 520-bp, about 530-bp, about 540-bp, about 550-bp, about 560-bp, about 570-bp, about 580-bp, about 590-bp, about 600-bp, about 610-bp, about 620-bp, about 630-bp, The sgRNAs target regions of the Oct4 promoter and / or enhancer region 90-bp, about 200-bp, about 300-bp, about 400-bp, about 500-bp, about 600-bp, about 700-bp, about 800-bp, about 900-bp, about 1000-bp, about 1500-bp, about 2000-bp, 2500-bp, about 3000-bp, 3500-bp, about 4000-bp, 4500-bp, about 5000-bp upstream. In some embodiments, any of the above sgRNAs target regions of the human Oct4 promoter and / or enhancer region.

[0039] In some embodiments, the sgRNA targets a region of the Sox2 promoter region about 1 to about 5000-bp upstream of the Sox2 transcription start site (TSS), about 100-bp to about 4000-bp upstream of the Sox2 TSS, about 200-bp to about 3000-bp upstream of the Sox2 TSS, about 400-bp to about 4000-bp upstream of the Sox2 TSS, about 500-bp to about 3000-bp upstream of the Sox2 TSS, about 500-bp to about 3000-bp upstream of the Sox2 TSS, about 600-bp to about 2000-bp upstream of the Sox2 TSS, or about 700-bp to about 1000-bp upstream of the Sox2 TSS. In some embodiments, the sgRNA is selected from the group consisting of about 10-bp, about 20-bp, about 30-bp, about 40-bp, about 50-bp, about 60-bp, about 70-bp, about 80-bp, about 90-bp, about 100-bp, about 110-bp, about 120-bp, about 130-bp, about 140-bp, about 150-bp, about 160-bp, about 170-bp, about 180-bp, about 190-bp, about 200-bp, about 210-bp, about 220-bp, about 230-bp, about 240-bp, about 250-bp, about 260-bp, about 270-bp, about 280-bp, about 290-bp, about 300-bp, about 310-bp, about 320-bp, about 330-bp, about 340-bp, about 350-bp, about 360-bp, about 370-bp, about 380-bp, about 390-bp, about 400-bp, about 410-bp, about 420-bp, about 430-bp, about 440-bp, about 450-bp, about 460-bp, about 470-bp, about 480-bp, about 490-bp, about 500-bp, about 510-bp, about 520-bp, about 530-bp, about 540-bp, about 550-bp, about 560-bp, about 570-bp, about 580-bp, about 590-bp, about 600-bp, about 610-bp, about 620-bp, about 630-bp, The sgRNA targets a region of about 1000-bp, about 1500-bp, about 2000-bp, about 2500-bp, about 3000-bp, about 3500-bp, about 4000-bp, 4500-bp, about 5000-bp upstream of the Sox2 promoter region. In some embodiments, any of the above sgRNAs targets a region of the human Sox2 promoter and / or enhancer region.

[0040] In some embodiments, the sgRNA targets a region of the Nanog promoter and / or enhancer region about 1 to about 5000-bp upstream of the Nanog transcription start site (TSS), about 100-bp to about 4000-bp upstream of the Nanog TSS, about 200-bp to about 3000-bp upstream of the Nanog TSS, about 400-bp to about 4000-bp upstream of the Nanog TSS, about 500-bp to about 3000-bp upstream of the Nanog TSS, about 500-bp to about 3000-bp upstream of the Nanog TSS, about 600-bp to about 2000-bp upstream of the Nanog TSS, or about 700-bp to about 1000-bp upstream of the Nanog TSS. In some embodiments, the sgRNA is selected from the group consisting of about 10-bp, about 20-bp, about 30-bp, about 40-bp, about 50-bp, about 60-bp, about 70-bp, about 80-bp, about 90-bp, about 100-bp, about 110-bp, about 120-bp, about 130-bp, about 140-bp, about 150-bp, about 160-bp, about 170-bp, about 180-bp, about 190-bp, about 200-bp, about 210-bp, about 220-bp, about 230-bp, about 240-bp, about 250-bp, about 260-bp, about 270-bp, about 280-bp, about 290-bp, about 300-bp, about 310-bp, about 320-bp, about 330-bp, about 340-bp, about 350-bp, about 360-bp, about 370-bp, about 380-bp, about 390-bp, about 400-bp, about 410-bp, about 420-bp, about 430-bp, about 440-bp, about 450-bp, about 460-bp, about 470-bp, about 480-bp, about 490-bp, about 500-bp, about 510-bp, about 520-bp, about 530-bp, about 540-bp, about 550-bp, about 560-bp, about 570-bp, about 580-bp, about 590-bp, about 600-bp, about 610-bp, about 620-bp, about 630-bp, Targets 90-bp, about 200-bp, about 300-bp, about 400-bp, about 500-bp, about 600-bp, about 700-bp, about 800-bp, about 900-bp, about 1000-bp, about 1500-bp, about 2000-bp, 2500-bp, about 3000-bp, 3500-bp, about 4000-bp, 4500-bp, about 5000-bp upstream of the Nanog promoter and / or enhancer region. In some embodiments, any of the above sgRNAs targets a region of the human Nanog promoter and / or enhancer region.

[0041] In some embodiments, the sgRNA targets a region of the Klf4 promoter and / or enhancer region about 1 to about 5000-bp upstream of the Klf4 transcription start site (TSS), about 100-bp to about 4000-bp upstream of the Klf4 TSS, about 200-bp to about 3000-bp upstream of the Klf4 TSS, about 400-bp to about 4000-bp upstream of the Klf4 TSS, about 500-bp to about 3000-bp upstream of the Klf4 TSS, about 500-bp to about 3000-bp upstream of the Klf4 TSS, about 600-bp to about 2000-bp upstream of the Klf4 TSS, or about 700-bp to about 1000-bp upstream of the Klf4 TSS. In some embodiments, the sgRNA is selected from the group consisting of about 10-bp, about 20-bp, about 30-bp, about 40-bp, about 50-bp, about 60-bp, about 70-bp, about 80-bp, about 90-bp, about 100-bp, about 110-bp, about 120-bp, about 130-bp, about 140-bp, about 150-bp, about 160-bp, about 170-bp, about 180-bp, about 190-bp, about 200-bp, about 210-bp, about 220-bp, about 230-bp, about 240-bp, about 250-bp, about 260-bp, about 270-bp, about 280-bp, about 290-bp, about 300-bp, about 310-bp, about 320-bp, about 330-bp, about 340-bp, about 350-bp, about 360-bp, about 370-bp, about 380-bp, about 390-bp, about 400-bp, about 410-bp, about 420-bp, about 430-bp, about 440-bp, about 450-bp, about 460-bp, about 470-bp, about 480-bp, about 490-bp, about 500-bp, about 510-bp, about 520-bp, about 530-bp, about 540-bp, about 550-bp, about 560-bp, about 570-bp, about 580-bp, about 590-bp, about 600-bp, about 610-bp, about 620-bp, about 630-bp, The sgRNAs target regions of the Klf4 promoter and / or enhancer region 90-bp, about 200-bp, about 300-bp, about 400-bp, about 500-bp, about 600-bp, about 700-bp, about 800-bp, about 900-bp, about 1000-bp, about 1500-bp, about 2000-bp, 2500-bp, about 3000-bp, 3500-bp, about 4000-bp, 4500-bp, about 5000-bp upstream. In some embodiments, any of the above sgRNAs target regions of the human Klf4 promoter and / or enhancer region.

[0042] In some embodiments, the sgRNA targets a region of the c-Myc promoter and / or enhancer region about 1 to about 5000-bp upstream of the c-Myc transcription start site (TSS), about 100 to about 4000-bp upstream of the c-Myc TSS, about 200-bp to about 3000-bp upstream of the c-Myc TSS, about 400-bp to about 4000-bp upstream of the c-Myc TSS, about 500-bp to about 3000-bp upstream of the c-Myc TSS, about 500-bp to about 3000-bp upstream of the c-Myc TSS, about 600-bp to about 2000-bp upstream of the c-Myc TSS, or about 700-bp to about 1000-bp upstream of the c-Myc TSS. In some embodiments, the sgRNA is selected from the group consisting of about 10-bp, about 20-bp, about 30-bp, about 40-bp, about 50-bp, about 60-bp, about 70-bp, about 80-bp, about 90-bp, about 100-bp, about 110-bp, about 120-bp, about 130-bp, about 140-bp, about 150-bp, about 160-bp, about 170-bp, about 180-bp, about 190-bp, about 200-bp, about 210-bp, about 220-bp, about 230-bp, about 240-bp, about 250-bp, about 260-bp, about 270-bp, about 280-bp, about 290-bp, about 300-bp, about 310-bp, about 320-bp, about 330-bp, about 340-bp, about 350-bp, about 360-bp, about 370-bp, about 380-bp, about 390-bp, about 400-bp, about 410-bp, about 420-bp, about 430-bp, about 440-bp, about 450-bp, about 460-bp, about 470-bp, about 480-bp, about 490-bp, about 500-bp, about 510-bp, about 520-bp, about 530-bp, about 540-bp, about 550-bp, about 560-bp, about 570-bp, about 580-bp, about 590-bp, about 600-bp, about 610-bp, about 620-bp, about 630-bp, The sgRNAs target 0-bp, about 200-bp, about 300-bp, about 400-bp, about 500-bp, about 600-bp, about 700-bp, about 800-bp, about 900-bp, about 1000-bp, about 1500-bp, about 2000-bp, 2500-bp, about 3000-bp, 3500-bp, about 4000-bp, 4500-bp, about 5000-bp upstream of the c-Myc promoter and / or enhancer region. In some embodiments, any of the above sgRNAs target the human c-Myc promoter and / or enhancer region.

[0043] In some embodiments, the sgRNA targets a region of the Nr5a2 promoter and / or enhancer region about 1 to about 5000-bp upstream of the Nr5a2 transcription start site (TSS), about 100-bp to about 4000-bp upstream of the Nr5a2 TSS, about 200-bp to about 3000-bp upstream of the Nr5a2 TSS, about 400-bp to about 4000-bp upstream of the Nr5a2 TSS, about 500-bp to about 3000-bp upstream of the Nr5a2 TSS, about 500-bp to about 3000-bp upstream of the Nr5a2 TSS, about 600-bp to about 2000-bp upstream of the Nr5a2 TSS, or about 700-bp to about 1000-bp upstream of the Nr5a2 TSS. In some embodiments, the sgRNA is selected from the group consisting of about 10-bp, about 20-bp, about 30-bp, about 40-bp, about 50-bp, about 60-bp, about 70-bp, about 80-bp, about 90-bp, about 100-bp, about 110-bp, about 120-bp, about 130-bp, about 140-bp, about 150-bp, about 160-bp, about 170-bp, about 180-bp, about 190-bp, about 200-bp, about 210-bp, about 220-bp, about 230-bp, about 240-bp, about 250-bp, about 260-bp, about 270-bp, about 280-bp, about 290-bp, about 300-bp, about 310-bp, about 320-bp, about 330-bp, about 340-bp, about 350-bp, about 360-bp, about 370-bp, about 380-bp, about 390-bp, about 400-bp, about 410-bp, about 420-bp, about 430-bp, about 440-bp, about 450-bp, about 460-bp, about 470-bp, about 480-bp, about 490-bp, about 500-bp, about 510-bp, about 520-bp, about 530-bp, about 540-bp, about 550-bp, about 560-bp, about 570-bp, about 580-bp, about 590-bp, about 600-bp, about 610-bp, about 620-bp, about 630-bp, The sgRNAs target regions of the Nr5a2 promoter and / or enhancer region 90-bp, about 200-bp, about 300-bp, about 400-bp, about 500-bp, about 600-bp, about 700-bp, about 800-bp, about 900-bp, about 1000-bp, about 1500-bp, about 2000-bp, 2500-bp, about 3000-bp, 3500-bp, about 4000-bp, 4500-bp, about 5000-bp upstream. In some embodiments, any of the above sgRNAs target regions of the human Nr5a2 promoter and / or enhancer region.

[0044] In some embodiments, the sgRNA targets a region of the Cebpa promoter and / or enhancer region about 1 to about 5000-bp upstream of the Cebpa transcription start site (TSS), about 100-bp to about 4000-bp upstream of the Cebpa TSS, about 200-bp to about 3000-bp upstream of the Cebpa TSS, about 400-bp to about 4000-bp upstream of the Cebpa TSS, about 500-bp to about 3000-bp upstream of the Cebpa TSS, about 500-bp to about 3000-bp upstream of the Cebpa TSS, about 600-bp to about 2000-bp upstream of the Cebpa TSS, or about 700-bp to about 1000-bp upstream of the Cebpa TSS. In some embodiments, the sgRNA is selected from the group consisting of about 10-bp, about 20-bp, about 30-bp, about 40-bp, about 50-bp, about 60-bp, about 70-bp, about 80-bp, about 90-bp, about 100-bp, about 110-bp, about 120-bp, about 130-bp, about 140-bp, about 150-bp, about 160-bp, about 170-bp, about 180-bp, about 190-bp, about 200-bp, about 210-bp, about 220-bp, about 230-bp, about 240-bp, about 250-bp, about 260-bp, about 270-bp, about 280-bp, about 290-bp, about 300-bp, about 310-bp, about 320-bp, about 330-bp, about 340-bp, about 350-bp, about 360-bp, about 370-bp, about 380-bp, about 390-bp, about 400-bp, about 410-bp, about 420-bp, about 430-bp, about 440-bp, about 450-bp, about 460-bp, about 470-bp, about 480-bp, about 490-bp, about 500-bp, about 510-bp, about 520-bp, about 530-bp, about 540-bp, about 550-bp, about 560-bp, about 570-bp, about 580-bp, about 590-bp, about 600-bp, about 610-bp, about 620-bp, about 630-bp, The sgRNAs target regions of the Cebpa promoter and / or enhancer region 90-bp, about 200-bp, about 300-bp, about 400-bp, about 500-bp, about 600-bp, about 700-bp, about 800-bp, about 900-bp, about 1000-bp, about 1500-bp, about 2000-bp, 2500-bp, about 3000-bp, 3500-bp, about 4000-bp, 4500-bp, about 5000-bp upstream. In some embodiments, any of the above sgRNAs target regions of the human Cebpa promoter and / or enhancer region.

[0045] In some embodiments, the sgRNA targets a region of the Esrrb promoter and / or enhancer region about 1 to about 5000-bp upstream of the Esrrb transcription start site (TSS), about 100-bp to about 4000-bp upstream of the Esrrb TSS, about 200-bp to about 3000-bp upstream of the Esrrb TSS, about 400-bp to about 4000-bp upstream of the Esrrb TSS, about 500-bp to about 3000-bp upstream of the Esrrb TSS, about 500-bp to about 3000-bp upstream of the Esrrb TSS, about 600-bp to about 2000-bp upstream of the Glis1 TSS, or about 700-bp to about 1000-bp upstream of the Esrrb TSS. In some embodiments, the sgRNA is selected from the group consisting of about 10-bp, about 20-bp, about 30-bp, about 40-bp, about 50-bp, about 60-bp, about 70-bp, about 80-bp, about 90-bp, about 100-bp, about 110-bp, about 120-bp, about 130-bp, about 140-bp, about 150-bp, about 160-bp, about 170-bp, about 180-bp, about 190-bp, about 200-bp, about 210-bp, about 220-bp, about 230-bp, about 240-bp, about 250-bp, about 260-bp, about 270-bp, about 280-bp, about 290-bp, about 300-bp, about 310-bp, about 320-bp, about 330-bp, about 340-bp, about 350-bp, about 360-bp, about 370-bp, about 380-bp, about 390-bp, about 400-bp, about 410-bp, about 420-bp, about 430-bp, about 440-bp, about 450-bp, about 460-bp, about 470-bp, about 480-bp, about 490-bp, about 500-bp, about 510-bp, about 520-bp, about 530-bp, about 540-bp, about 550-bp, about 560-bp, about 570-bp, about 580-bp, about 590-bp, about 600-bp, about 610-bp, about 620-bp, about 630-bp, Targets 90-bp, about 200-bp, about 300-bp, about 400-bp, about 500-bp, about 600-bp, about 700-bp, about 800-bp, about 900-bp, about 1000-bp, about 1500-bp, about 2000-bp, 2500-bp, about 3000-bp, 3500-bp, about 4000-bp, 4500-bp, about 5000-bp upstream of the Esrrb promoter and / or enhancer region. In some embodiments, any of the above sgRNAs targets a region of the human Esrrb promoter and / or enhancer region.

[0046] In some embodiments, the sgRNA targets a region of the Rex1 promoter and / or enhancer region about 1 to about 5000-bp upstream of the Rex1 transcription start site (TSS), about 100-bp to about 4000-bp upstream of the Rex1 TSS, about 200-bp to about 3000-bp upstream of the Rex1 TSS, about 400-bp to about 4000-bp upstream of the Rex1 TSS, about 500-bp to about 3000-bp upstream of the Rex1 TSS, about 500-bp to about 3000-bp upstream of the Rex1 TSS, about 600-bp to about 2000-bp upstream of the Rex1 TSS, or about 700-bp to about 1000-bp upstream of the Rex1 TSS. In some embodiments, the sgRNA is selected from the group consisting of about 10-bp, about 20-bp, about 30-bp, about 40-bp, about 50-bp, about 60-bp, about 70-bp, about 80-bp, about 90-bp, about 100-bp, about 110-bp, about 120-bp, about 130-bp, about 140-bp, about 150-bp, about 160-bp, about 170-bp, about 180-bp, about 190-bp, about 200-bp, about 210-bp, about 220-bp, about 230-bp, about 240-bp, about 250-bp, about 260-bp, about 270-bp, about 280-bp, about 290-bp, about 300-bp, about 310-bp, about 320-bp, about 330-bp, about 340-bp, about 350-bp, about 360-bp, about 370-bp, about 380-bp, about 390-bp, about 400-bp, about 410-bp, about 420-bp, about 430-bp, about 440-bp, about 450-bp, about 460-bp, about 470-bp, about 480-bp, about 490-bp, about 500-bp, about 510-bp, about 520-bp, about 530-bp, about 540-bp, about 550-bp, about 560-bp, about 570-bp, about 580-bp, about 590-bp, about 600-bp, about 610-bp, about 620-bp, about 630-bp, Targeting regions 90-bp, about 200-bp, about 300-bp, about 400-bp, about 500-bp, about 600-bp, about 700-bp, about 800-bp, about 900-bp, about 1000-bp, about 1500-bp, about 2000-bp, 2500-bp, about 3000-bp, 3500-bp, about 4000-bp, 4500-bp, about 5000-bp upstream of the Rex1 promoter and / or enhancer region.

[0047] In some embodiments, the Oct4 promoter targeted by the sgRNA is selected from SEQ ID NOs: 1-6, 57, and 58. In some embodiments, the Oct4 enhancer targeted by the sgRNA is selected from SEQ ID NOs: 7-11. In some embodiments, the Sox2 promoter targeted by the sgRNA is selected from SEQ ID NOs: 12-21 and 59. In some embodiments, the Klf4 gene targeted by the sgRNA is selected from SEQ ID NOs: 22-31 and 60. In other embodiments, the c-Myc gene targeted by the sgRNA is selected from SEQ ID NOs: 32-41 and 61. In other embodiments, the Nr5a2 gene targeted by the sgRNA is selected from SEQ ID NOs: 42-45. In other embodiments, the Glis1 gene targeted by the sgRNA is selected from SEQ ID NOs: 46-50. In other embodiments, the Cebpa gene targeted by the sgRNA is selected from SEQ ID NOs: 51-56. In other embodiments, the Lin28 gene targeted by the sgRNA comprises SEQ ID NO: 62. In other embodiments, the Nanog gene targeting sgRNA comprises SEQ ID NO: 63. In another embodiment, the EEA motif targeting sgRNA comprises SEQ ID NO: 64, where the EEA motif is a regulatory region associated with transcription of pluripotency genes, including but not limited to, a PRD-like homeodomain TR binding site.

[0048] IV.CRISPR activation system The present disclosure is based on a CRISPR activation system derived from the bacterial CRISPR / Cas system. The CRISPR activation system comprises a type II Cas with inactivated nuclease activity and fused to an effector to regulate gene transcription. In some embodiments, the dCas is fused to at least one transcription activator. In some embodiments, the dCas is fused to a tandem array of peptides that serves as a scaffolding structure to link the dCas to multiple transcription activators. In some embodiments, the tandem array of peptides is a SunTag array. In other embodiments, the transactivator is a tetramer of herpes simplex VP16 transcription activator domain (VP64). In one embodiment, the CRISPR activation system is dCas-SunTag-VP64, also known as the SunTag CRISPR activation system.

[0049] In some embodiments, the CRISPR activation system comprises dCas9 fused with SunTag array, and at least one acetyltransferase active domain of p300 (p300 core) bound to SunTag array.In other embodiments, the method for generating iPSC comprises using the CRISPR activation system comprising dCas9, SunTag array, and p300 core to modify the histone acetylation of endogenous locus, thus regulating the transcription of gene.It is understood that other CRISPR activation systems and / or transactivators known to those skilled in the art can be used to generate iPSC. EXAMPLES

[0050] The following examples are intended to further illustrate certain embodiments of the present disclosure. The examples are presented to assist those of ordinary skill in the art and are not intended to limit the scope thereof.

[0051] Example 1. Activation of endogenous Oct4 and Sox2 by dCas9-SunTag-VP64 To determine whether and how reconstitution of endogenous loci initiates reprogramming toward pluripotency, we used the SunTag CRISPR activation system to precisely reconstitute endogenous pluripotency loci in mouse embryonic fibroblasts (MEFs). dCas9-SunTag-VP64 was selected to enhance its chromatin remodeling activity by recruiting multiple VP64s to one targeted site (Figure 1A) (Tanenbaum et al., 2014). Expression of dCas9 was controlled by the Tet-On promoter. The Oct4 and Sox2 loci were selected as targets due to their central roles in inducing and maintaining pluripotency.

[0052] MEFs were prepared from E13.5 mouse embryos. After recovery of the embryos, the head, limbs, and internal organs, especially the gonads, were removed under a dissecting microscope. The remaining body of the embryo was minced with two blades and digested with 0.05% trypsin-EDTA for 15 min. Then, MEF medium was added to stop the trypsinization. Further separation of tissues was performed by pipetting up and down several times. Then, the cells were collected by centrifugation and plated in 15 cm dishes for expansion (P0). MEF cells were used before P3 in all tests and were cultured in DMEM supplemented with 10% FBS and non-essential amino acids.

[0053] sgRNAs were designed to target the Oct4 and Sox2 promoters, as well as the Oct4 enhancer. In addition to the activating effect of sgRNAs, multiple factors were considered for the targeted genomic sequences, including proximity to but not overlapping with binding sites for pluripotency factors and transcription machinery, histone H3K27 acetylation in pluripotent stem cells, and their potential to form promoter-enhancer loops mediated by the Mediator complex (Figures 5A-5C).

[0054] For sgRNA constructs, 72-bp oligos containing specific sgRNAs were synthesized for PCR using primers sgRNA-F (SEQ ID NO: 173-GTATCCCTTGGAGAACCACCT) and sgRNA-R (SEQ ID NO: 174--TGCTGTTTCCAGCTTAGCTCT). The amplified fragments were purified and used for recombination reactions with pSLQ1373 constructs digested with BstXI and BlpI according to the Gibson Assembly Cloning Kit protocol (NEB).

[0055] The level of transcriptional activation of target genes was examined using each designed Oct4 and Sox2 sgRNA delivered by lentivirus in differentiating mouse embryonic stem (ES) cells (Figure 5D). HEK293T / 17 cells (ATCC® CRL-11268) were cultured in DMEM supplemented with 10% FBS and plated one day in advance to reach approximately 70% confluency for transfection, and VSV-G envelope expression plasmids pMD2.G (Addgene, 12259) and psPAX2 (Addgene, 12260) were used for lentivirus packaging. Plasmids containing the gene of interest (1.8 μg) were mixed with psPAX2 (1.35 μg) and pMD2.G (0.45 μg) in each well of a 6-well plate, and 10.8 μl of FUGENE HD (Promega) was added for transfection. After 5 hours, the medium was changed. Virus-containing supernatants were harvested at 48 h, passed through a 0.45 μM filter to remove cell debris, and mixed with one volume of fresh medium for immediate use. For the SunTag system, the three components (dCas9, VP64, and Tre3G) of lentivirus were packaged separately and mixed at the time of use. SunTag transduction was performed with two rounds of lentiviral infection: the first round with the Suntag system (dCas9, VP64, and Tre3G) and the second round with sgRNA. Medium was changed after each infection.

[0056] Mouse ES cells were first transduced with the dCas9-SunTag-VP64 system and sgRNA. Because Oct4 and Sox2 are highly expressed in ES cells, ES cells were induced to differentiate in MEF medium supplemented with 1 μM retinoic acid for 4 days, which reduced the expression of Oct4 and Sox2. Meanwhile, the dCas9-SunTag-VP64 system was induced with doxycycline. Analysis of Oct4 expression showed that sgRNAs targeting a narrow promoter region close to the transcription start site (TSS) and a 200-bp region of the distal enhancer could increase transcription (Figure 5E). As for the Sox2 promoter, sgRNA activity showed a significant trend of higher gene activation with sgRNAs closer to the TSS (Figure 5E).

[0057] The selected sgRNAs and dCas9-SunTag-VP64 were also transduced into MEFs (Figure 5F). MEF cells were incubated for 8 h or overnight in the presence of lentiviral supernatant and 5 μg / ml polybrene (Millipore). sgRNAs O-127 and O-71, targeting 127- and 71-bp upstream of the Oct4 TSS, were combined to target the promoter. Similarly, O-1965, O-2066, and O-2135 were combined to target the Oct4 enhancer, and individually S-84, S-136, and S-148 were combined to target the Sox2 promoter. Four days after dCas9 induction with doxycycline, targeting the Oct4 promoter increased Oct4 transcription by approximately 100-fold, while targeting the enhancer resulted in modest activation (Figure 5G). For the Sox2 promoter, approximately 15-fold activation was detected (Figure 5G). This suggests that guided by specific sgRNAs, dCas9-SunTag-VP64 can activate the silenced endogenous Oct4 and Sox2 genes in MEFs.

[0058] Viral titration was performed in primary MEF cells by detecting blue fluorescent protein (BFP) from the sgRNA cassette, allowing calculation of the multiplicity of infection (MOI) using a Poisson distribution (Arai et al., 1999). P(k)=e -m m k / k! where m is the MOI, k is the number of viral particles, and P(k) is the percentage of cells infected with k viral particles. In the disclosed experiment, approximately 70% of MEFs were positive for BFP, and the MOI was 1.20. Similarly, the percentage of cells infected with the indicated number of sgRNA viral particles is: [Table 1]

[0059] Example 2. Establishment of pluripotency network in MEFs by gene activation with dCas9-SunTag-VP64 We next investigated whether the pluripotency network could be fully reactivated and established in MEFs. The SunTag reprogramming system was optimized in two ways. First, more genes were targeted by adding the corresponding sgRNAs. Klf4, c-Myc (Takahashi and Yamanaka, 2006), Nr5a2 (Heng et al., 2010), Glis1 (Maekawa et al., 2011), and Cebpa (Di Stefano et al., 2014) were selected. For each promoter, 4–10 sgRNAs were designed and tested in differentiating ES cells (Figure 5E). One to three sgRNAs for each promoter were included in the previous Oct4 / Sox2 sgRNA pool (see Table 1). Next, a small molecule cocktail consisting of Parnate, Chir99021, A83-01, and forskolin (PCAF) was added to the reprogramming medium. This chemical cocktail further increased Oct4 and Sox2 transcription by 3-4 fold at day 4 (Figure 5H). [Table 2-1] [Table 2-2]

[0060] To monitor the reactivation of the pluripotency network, MEF cells from OG2 mice (B6;CBA-Tg(Pou5f1-EGFP)2Mnn / J, Jackson Laboratory) were used at E13.5. OG2 MEFs harbor a stable Oct4-EGFP reporter and show a strong EGFP signal when endogenous Oct4 is actively transcribed (Szabo et al., 2002). 24 h prior to transduction, MEF cells were plated at 10,000 cells / cm. 2 Cells were seeded onto gelatin-coated plates at a density of 10,000 cells / cm. After transduction with dCas9-SunTag-VP64 and sgRNA pools (18 sgRNAs in total, see Table 1), cells were allowed to recover in MEF medium for 24 h. To initiate reprogramming, MEF medium was changed to reprogramming medium (ES medium supplemented with 10 μM parnate, 3 μM Chir99021, 1 μM A83-01, and 10 μM forskolin) supplemented with 1 μg / ml doxycycline. This was referred to as day 0 (Figure 1B). On day 3, cells were treated with 1 mg / ml collagenase B (Roche) for 20 min, followed by 0.05% trypsin for 5 min at 37 °C and plated in new wells (30,000 cells / cm). 2 ) and further cultured until the end of reprogramming. From day 4, Oct4 and Sox2 transcription became increasingly robust (Figure 1D). By day 7, reprogramming clusters appeared, and after 2 weeks, EGFP-positive colonies were seen (Figure 1C). These colonies were also positive for Nanog, Sox2, and SSEA-1 (Figure 1F). Throughout the process, the medium was changed every other day for the first 12 days. After that, regular ES medium was used and changed every day, and EGFP-positive colonies were usually ready for iPSC derivation between days 16 and 18.

[0061] EGFP-positive colonies were then expanded on feeder cells to generate CRISPR iPSC lines. For iPSC induction, the reprogramming cultures were incubated with 1 mg / ml collagenase B (Roche) for 20 min at 37 °C. Single colonies were picked up under a microscope and digested with 0.05% trypsin for 5–10 min into a single-cell suspension. The cells were then plated onto feeders in regular ES medium, and these cells are considered as P0 iPSCs. CRISPR iPSCs formed typical mouse ES-like dome-shaped colonies with strong EGFP signals (Figure 1E). A panel of pluripotency genes, including Oct4, Sox2, Nanog, Esrrb, Nr5a2, and Utf1, were highly expressed (Figure 1G). These cells could be passaged for more than 20 passages without any signs of losing EGFP signal or ES morphology (Figure 1E). These data indicate that pluripotency is established in these CRISPR iPSCs.

[0062] All iPSC lines were maintained on feeders in KO-DMEM (Invitrogen) with 5% ES-FBS (Invitrogen) and 15% KO-serum replacement (Invitrogen), 1% GlutaMAX™ (Invitrogen), 1% non-essential amino acids (Invitrogen), 55 μM 2-mercaptoethanol (Sigma), 10 ng / ml leukemia inhibitory factor (Stemgent), 3 μM CHIR99021, and 1 μM PD0325901.

[0063] Example 3. Single gene coordinate targeting of Sox2 gene to establish CRISPR iPSCs To identify essential loci required for CRISPR iPSC generation, sgRNAs targeting individual loci were removed one by one from the pool. In this 18-sgRNA pool, removal of sgRNAs targeting the Oct4, Sox2, or Glis1 promoters or the Oct4 enhancer led to a sharp decrease in the number of EGFP-positive colonies (Figure 6A), indicating a potential role for these loci in pluripotency induction.

[0064] Next, it was determined whether targeting Oct4, Sox2, and Glis1 promoters together was sufficient to generate iPSCs. Because a single sgRNA targeting each gene could achieve 60% to 180% gene activation compared to a combination of two or three sgRNAs targeting the same genes (Figure 6B), one sgRNA was selected targeting the promoter of each gene: O-127 for Oct4, S-84 for Sox2, and G-215 for Glis1. This approach could simplify the reprogramming system and reduce off-target effects. The combination of OSGs (O-127, S-84, and G-215) could adequately activate the three genes (Figure 6C). After two weeks, EGFP-positive colonies were observed, and iPSC lines could be established (Figure 6D and 6E).

[0065] During OSG reprogramming, it was surprisingly noticed that EGFP-positive colonies appeared when only S-84 was used (Figure 6F), suggesting that targeting only the Sox2 promoter may be sufficient for pluripotency induction. To exclude any possible off-target effects of S-84, the top 10 predicted targets of S-84 were examined. The off-targets of sgRNAs were predicted by CCTop-CRISPR / Cas9 target online predictor (Stemmer et al., 2015). In each prediction, the core sequence was set to 12 bp. The maximum mismatch of the core sequence was 2 bp, and the maximum mismatch of all mismatches was 4 bp. For S-84, only the Sox2 gene was significantly activated (Figure 6G). Sox2 protein was also detected on day 4 (Figure 2B). Furthermore, the reprogramming procedure was repeated using another two Sox2-targeting sgRNAs, S-136 and S-148 (Figure 2A). These two sgRNAs individually activated endogenous Sox2 transcription (Figure 2C), resulting in EGFP-positive colonies (Figure 2D).

[0066] We next investigated the authenticity of the pluripotency of these iPSCs. Nanog and SSEA-1 proteins were also detected within these EGFP-positive colonies, and CRISPR iPSC lines were established (Figure 2E and 2F). For line S-17, expression of key pluripotency factors was similar to that in R1 mouse ES (R1 ES) cells (Figure 2G). Karyotype analysis of the iPSC lines was performed at Cell Line Genetics, and Giemsa binding was analyzed. The generated iPSCs were also karyotypically normal (Figure 2H).

[0067] S-17 cells were B6 albino (B6(Cg)-Tyr c-2J A more rigorous assay of pluripotency was performed in which S-17 cells were injected into blastocysts of a 100% (1 / J, Jackson Labs) background. EGFP-positive cells were found in the gonadal region of 71.4% (5 out of 7) of E13.5 embryos (Figure 2I). Productive chimeras were generated at a rate of 46.2% (Figure 2J) (6 out of 13). More importantly, B6 albino female mice (6 weeks) were used to mate with chimeric mice (4-8 weeks) for germline transmission studies, and S-17 cells were also germline-competent (Figure 2K). These data concluded that monogenic targeting of the Sox2 promoter by a single sgRNA is sufficient to reprogram MEFs into bona fide pluripotent stem cells.

[0068] R1 ES cells were maintained on feeders in KO-DMEM (Invitrogen) containing 5% ES-FBS (Invitrogen) and 15% KO-serum replacement (Invitrogen), 1% GlutaMAX™ (Invitrogen), 1% non-essential amino acids (Invitrogen), 55 μM 2-mercaptoethanol (Sigma), 10 ng / ml leukemia inhibitory factor (Stemgent), 3 μM CHIR99021, and 1 μM PD0325901. For microinjection, iPSCs were maintained under feeder-free N2B27 conditions (50% DMEM / F12, 50% Neurobasal medium, 0.5% N2 medium, 1% B27 medium, 0.1 mM 2-mercaptoethanol, 10 ng / ml leukemia inhibitory factor, 25 μg / ml BSA, 3 μM CHIR99021, and 1 μM PD0325901). On the day of injection, cells were suspended in blastocyst injection medium (25 mM HEPES-buffered DMEM + 10% FBS, pH 7.4).

[0069] For the generation of chimeric mice and germline transmission studies, 4-week-old superovulated B6(Cg)-Tyr c-2J / J(B6-albino) mice (Jackson Laboratory) were cultured at 4°C for blastocyst preparation using B6(Cg)-Tyr c-2J The blastocysts were mated with B6(Cg)-Tyr / J male mice. Morulae (2.5 days post-coitum) were collected and cultured overnight at 37°C and 5% CO2 in KSOM medium (Millipore). The following morning, the blastocysts were ready for iPSC injection, with approximately 10–20 cells injected per blastocyst. The injected blastocysts were cultured in KSOM medium at 37°C and 5% CO2 for 1–2 h and then transferred into the uterus of pseudopregnant CD1 female mice (Charles River, stock #022) 2.5 days post-coitum. Chimeric mice can be identified by the color of their mosaic coat. Male chimeric mice were further bred with female B6(Cg)-Tyr c-2J / J mice, and offspring with black coat color are considered successful germline transmission. For gonadal contribution, injected embryos were allowed to recover 10 days after implantation (E13.5). The gonadal region of each embryo was collected and EGFP signal was visualized under a microscope. All animal procedures were approved by the Institutional Animal Care and Use Committee of the University of California, San Francisco.

[0070] Example 4. S-17 MEFs are reprogrammable, highly efficient, and low variability Reprogramming efficiency by lentiviral transduction was relatively low and variable in both OG2 and 129 mice (129S2 / SvPasCrl, Charles River) and derived MEFs (0-0.013%) (Figures 2C, 6A, 6F, 7A-7C). This may be due to inefficient delivery of the SunTag components and random copy numbers of the components delivered to the cells. This is reflected by the variable copy numbers of the sgRNA cassette in the genome of established iPSC lines, where 1-5 copies per cell were found among 12 lines (Figure 7D). For quantification of the sgRNA cassette in the genome, quantitative RT-PCR (qPCR) primers were designed for amplification of the Sox2 gene and sgRNA cassette in the genome (see Table 2). Amplification of Sox2 was used to normalize the genome of each cell line. Plasmids containing the target were used to generate a standard curve. A standard curve was constructed by plotting the Ct values ​​against the plasmid copy numbers for a series of plasmid dilutions (10 pg, 1 pg, 0.1 pg, 0.01 pg) and the copy numbers of the Sox2 gene and sgRNA cassette in approximately 30 ng of genomic DNA were calculated based on the standard curve. The sgRNA copy numbers were then calculated by normalizing to the Sox2 gene (2 copies / cell). [Table 3-1] [Table 3-2] [Table 3-3]

[0071] To reduce variability and increase efficiency, secondary MEFs were generated using CRISPR iPSC lines derived from a single colony. S-17 iPSCs were labeled with blue fluorescent protein (BFP) and injected into B6 blastocysts, and secondary MEFs were derived from E13.5 embryos (Figure 3A). Approximately half (52.4%) of the MEFs were revealed by flow cytometry to be derived from S-17 iPSCs (Figure 7E). During this flow cytometry, cells were trypsinized to form a suspension of single cells in FACS buffer, and this suspension was filtered through a 40 μm cell strainer before examination on a MACSQuant VYB flow cytometer. Data were analyzed with FlowJo v10. These derived secondary MEFs were referred to as S-17 MEFs. Upon doxycycline induction, endogenous Sox2 was readily detected by both qPCR and immunofluorescence staining, and Sox2 was undetectable in the absence of doxycycline (Figure 3B and 3D). Off-target genes were not significantly elevated (Figure 7G). These data indicate that the SunTag system functioned properly to activate Sox2 in S-17 MEFs.

[0072] We next investigated whether S-17 MEFs could be reprogrammed. Sox2 transcription was significantly upregulated at day 4 and rapidly increased to levels comparable to R1 ES cells by day 8 (Figure 3B). Following the upregulation of Sox2, other core pluripotency factors Oct4, Nanog, and Rex1 were also activated. Their transcription was detected at day 8 and dramatically elevated thereafter (Figure 3G). Meanwhile, morphological changes were observed from day 4, and EGFP-positive colonies were visible at day 7 (Figure 3E). iPSC lines could also be established (Figure 3E). Using S-17 MEFs, reprogramming efficiency (0.1%) increased 40-fold compared to the lentiviral method (Figure 3F). As expected, much less variability was observed (Figure 7H).

[0073] For qPCR (see Table 2), total RNA was extracted from samples at the indicated times using RNeasy Plus mini kit with QiaShredder (Qiagen) and treated with DNA-free kit (Ambion) to remove genomic DNA. RNA was reverse transcribed using iScript cDNA synthesis kit (Bio-Rad). Quantitative PCR was performed using iQTM SYBR Green Supermix (Bio-Rad) on a 7500Fast Real-Time PCR System (Applied Biosystems). All reactions were performed in quadruplicate. All data were statistically analyzed in Prism7. For immunofluorescence staining, cells were washed three times with DPBS and fixed with 4% PFA for 30 min at 4 °C. Donkey serum (10% in DPBS) was used for blocking for 1 h at 4 °C. Antibodies were diluted in DPBS containing 1% BSA. The following primary antibodies were used for staining: anti-Sox2 (1:1000, Millipore, AB5603), anti-Oct4 (1:1000, Santa Cruz, sc-5279), anti-Nanog (1:500, Abcam, 80892), and anti-SSEA-1 (1:200, Stemgent, 09-0095).

[0074] Further differentiated tail tip fibroblasts (TTFs) were also tested whether they could be reprogrammed. S-17 TTFs were derived from 14-month-old adult chimeric mice. Tails were stripped, minced into 1 mm pieces, and cultured in 6 cm dishes. The medium was half-changed every 3 days until fibroblasts migrated out of the grafts. Induced TTFs were maintained in DMEM supplemented with 10% FBS and non-essential amino acids and passaged ready for use (P1). In the presence of doxycycline, TTFs underwent morphological changes and EGFP-positive colonies were obtained in 2 weeks (Figure 7L). These observations indicate that both S-17 MEFs and TTFs can be reprogrammed.

[0075] For reprogramming with S-17 MEFs or mouse TTFs, plate MEFs or TTFs at 5,000 cells / cm on gelatin-coated plates. 2The cells were seeded at a density of 100 x 1000 cells / well. After 24 hours, the medium was switched to reprogramming medium containing 1 μg / ml doxycycline, which was indicated as day 0. The medium was changed every other day until day 14. EGFP-positive colonies were counted for calculation of reprogramming efficiency or used to derive iPSC lines.

[0076] Example 5. Reconstitution of the Sox2 promoter triggers reprogramming of S-17 MEFs towards pluripotency Without doxycycline, Sox2 activation was undetectable and no colonies were obtained (Figures 3B and 3J). Removal of the PCAF cocktail from the medium still generated EGFP-positive colonies, albeit with reduced efficiency (Figure 7I). This observation led us to conclude that endogenous Sox2 activation is the trigger for S-17 MEF reprogramming.

[0077] We next examined whether reprogramming was dose-dependent on Sox2 levels. Sox2 was activated at a range of doxycycline concentrations, e.g., 0, 0.01, 0.1, and 1 μg / ml. Sox2 levels were positively correlated with doxycycline concentrations, and reprogramming efficiency was clearly dependent on Sox2 levels (Figure 3J).

[0078] Because VP64 promotes gene transcription and chromatin remodeling by recruiting multiple epigenetic modifiers (Hirai et al., 2010), we investigated whether and how the SunTag system could epigenetically remodel the Sox2 promoter. Chromatin immunoprecipitation (ChIP) was performed using an H3K27 acetylation (H3K27ac) antibody against the Sox2 promoter. All ChIP experiments were performed using the EZ-ChIP chromatin immunoprecipitation kit (Millipore, 17-371) according to the protocol provided with the kit, with modifications. Briefly, approximately 2 × 10 6Cells were crosslinked with 0.275 ml of 37% formaldehyde in 10 ml of growth medium. Unreacted formaldehyde was quenched by adding 1 ml of 1.25 M glycine (10x). 0.12 ml of SDS lysis buffer was used for each sample. Then, genomic DNA was sheared to lengths of 100–500 bp in a Covaris S2 Sonicator with optimized conditions. 1.5 μg of H3K27 acetylation antibody (Abcam, ab4729) and 15 μL of magnetic protein A / G beads (Millipore 16-663) were used for each sample. Finally, DNA fragments were eluted with 50 μl of elution buffer C and used for downstream qPCR.

[0079] As early as day 4, H3K27ac levels were already elevated two-fold and further increased at days 8 and 12 (Figure 3C). This indicates that the SunTag system targeting Sox2 triggered a gradual and constant epigenetic remodeling at the Sox2 promoter. Epigenetic remodeling of the promoters of Oct4, Nanog, and Rex1 was also tested, and their H3K27ac levels, as well as their gene transcription, were significantly increased at a latency of 4 days (Figures 3G and 3H). Interestingly, the enhancer of Oct4 showed a concomitant elevation of H3K27ac levels (Figure 3I). These data suggest that Sox2 activation promoted the subsequent induction of other important genes for the establishment of pluripotency.

[0080] We next tested whether additional targeting of the Oct4 promoter in S-17 MEFs promoted reprogramming efficiency. Transduction of O-127 led to a significant increase in Oct4 transcription, which also increased reprogramming efficiency (Figure 3K). This synergistic effect supported the idea that Oct4 and Sox2 cooperated in inducing pluripotency.

[0081] We also compared S-17 MEF reprogramming with conventional reprogramming using overexpressed factors. Unlike S-17 MEFs, overexpressed factors failed to epigenetically reconstitute the Sox2 promoter at day 4 (Figure 7J), and Sox2 transcription at the endogenous locus was effectively undetectable at days 4 and 12 (Figure 3L). After 3 weeks, overexpression of Oct4 or Sox2 failed to generate colonies, while overexpression of Oct4, Sox2, and Klf4 (OSK) generated slightly more EGFP-positive colonies than S-17 MEFs, with greater variability between experiments (Figures 3M and 7K).

[0082] Example 6. Simultaneous reconstitution of Oct4 promoter and enhancer reprograms MEFs into iPSCs Reconstitution of both the Oct4 promoter and enhancer was critical for pluripotency induction, and targeting of the promoter alone was insufficient for the generation of EGFP-positive colonies (Fig. 6A and 2C). Given that key pluripotency factors as well as p300 and Mediator complexes are enriched at the Oct4 distal enhancer in mouse ES cells (Fig. 4A), it was hypothesized that simultaneous reconstitution of the Oct4 promoter and enhancer is required for pluripotency induction.

[0083] To test the hypothesis, we used a dual-sgRNA cassette that transcribes two sgRNAs targeting different sites (Figure 8A). The O-127-2066 cassette targeted the Oct4 promoter (O-127) and enhancer (O-2066) at the single-cell level. To generate the dual-sgRNA construct, a fragment containing the second sgRNA was amplified with the single-sgRNA construct as a template using primers mU6-T2H-F (sequence number 167 ctaggatccattaggcGGGTACAGTGCAGGGGAA) and mU6-T2H-R2 (sequence number 168 atacggttatccacgcGGCCGCCTAATGGATCCT). This fragment was purified and used in a recombination reaction with the other construct containing the first sgRNA digested with NotI. The second mU6-sgRNA cassette is downstream of the first cassette in the same transcriptional direction.

[0084] O-127-2066 simultaneously reconstituted the Oct4 promoter and enhancer with elevated levels of H3K27ac (Figure 4B). The levels of gene transcription by O-127-2066 were similar to O-127 at days 4 and 8 (Figure 4C). However, from day 8 onwards, Oct4 transcription was further elevated in O-127-2066 cultures. Notably, when cells were re-plated at days 7 and 11 to allow cell expansion, overall Oct4 expression in the population increased dramatically (Figure 4C). In O-127 and O-2066 cultures, the weak Oct4 expression remained almost unchanged after 8 days (Figure 4C). Thus, by day 12, EGFP-positive colonies were observed in O-127-2066 cultures, and those colonies also expressed Nanog, Sox2, and SSEA-1, indicating an acquired core pluripotency network (Figures 4E and 4F). iPSC lines could be derived from these colonies (Figure 4E), whereas no colonies were observed in O-127 or O-2066 cultures (Figure 4D).

[0085] Potential off-target activation was examined. When the top 10 predicted targets of sgRNAs O-127 and O-2066 were examined, no significant activation of off-target genes was detected (Figure 8C). Another two dual sgRNA cassettes, O-127-1965 and O-127-2135, were also tested in parallel, and similar results were observed (Figure 4D). These data strongly supported that pluripotency is induced by simultaneous reconstitution of the endogenous Oct4 promoter and enhancer.

[0086] A bona fide pluripotent stem cell line was also achieved. The D-9 line showed expression of pluripotency genes similar to R1 ES cells and a normal karyotype (Figures 4G and 4H). After injection of D-9 cells into B-6 albino blastocysts, live chimeric mice were generated among the offspring at a rate of 60% (6 out of 10) (Figure 4J). This line contributed significantly to the gonadal region of 75% E13.5 embryos (6 out of 8) (Figure 4I), and germline transmission was confirmed in 50% of the offspring (2 out of 4) (Figure 4K).

[0087] Example 7. Epigenetic remodeling of histone acetylation at the Oct4 promoter and enhancer reprograms MEFs into iPS cells Chromatin remodeling by VP64 is due to its primary function in recruiting the transcription machinery. To more rigorously determine whether epigenetic remodeling is sufficient to initiate reprogramming, histone acetylation of the Oct4 promoter and enhancer was increased by specific manipulation. Histone acetylation was manipulated because acetylation of histone H3K27 simultaneously marks the Oct4 promoter and enhancer regions (Figure 4A). p300 core possesses only the acetyltransferase activity domain of p300 and was proven to enhance the acetylation of targeted histones (Hilton et al., 2015). Therefore, VP64 was replaced with p300 core to generate the dCas9-SunTag-p300 core system (Figure 8E).

[0088] For cloning of p300 core, the backbone was obtained from pSLQ1711-pPGK-ScFV(GCN4)-sfGFP-VP64 by digestion with SbfI-HF and RsrII (NEB) and gel purification kit (Qiagen). An 83-bp SV40 nuclear localization site (NLS) with a linker was cloned and added between sfGFP and p300 core with forward primer (SEQ ID NO: 169 TACAAAGGTGGAGGTCGGACCGaaggcagcggctcccccaag) and reverse primer (SEQ ID NO: 170 AAATCGTCTAAAGCATCcgaccctccgccggaaccgccca). The p300 core was PCR amplified from template pcDNA-dCas9-p300 core (Addgene 61357) using Phusion® High-Fidelity DNA Polymerase (NEB) with forward primer (SEQ ID NO: 171 AGTGGGCGGTTCCGGCGGAGGGTCGattttcaaaccagaagaactacgac) and reverse primer (SEQ ID NO: 172 TATCAAGCTTGCATGCCTGCAGGTTAgtcctggctctgcgtgtgcagctc). The backbone, including the 83-bp SV4 NLS and linker, and the p300 core were then assembled using the Gibson Assembly Cloning Kit (NEB).

[0089] Reprogramming experiments were performed with the dCas9-SunTag-p300 core system. Although p300 core cultures showed comparable H3K27ac levels at the Oct4 promoter and enhancer as VP64, only 1 / 30 of Oct4 transcription was detected in p300 core cultures at day 5 (Figure 8F and 8G). This could be explained by the inability of p300 core to recruit the transcription machinery. Cultures were then passaged on days 9 and 14. Interestingly, by day 10, Oct4 levels were comparable in VP64 and p300 core cultures (Figure 8G). Accordingly, EGFP-positive colonies were generated in p300 core cultures and iPSC lines were generated (Figure 8H and 8I). These observations indicate that manipulation of histone acetylation by p300 core led to chromatin remodeling similar to VP64, although with a notable latency in transcription activation. Together, these results indicate that epigenetic remodeling of the Oct4 promoter and enhancer via VP64 or p300 core is sufficient to trigger reprogramming toward pluripotency.

[0090] Due to the widespread binding of ectopic proteins, it is very challenging to identify the remodeling events on endogenous chromatin that trigger reprogramming towards pluripotency. To gain insight into this, we precisely remodeled specific sites on endogenous chromatin using the CRISPRa system, dCas9-SugTag-VP64 or dCas9-SunTag-p300 cores. For the first time, we found that targeted remodeling of a single gene, either Oct4 or Sox2, was sufficient to initiate reprogramming towards pluripotency, and a bona fide pluripotent stem cell lineage was established. CRISPR iPSC-derived S-17 MEFs and TTFs were also reprogrammable, suggesting that activation of endogenous Sox2 is a key event for pluripotency induction. Simultaneous remodeling of the promoter and enhancer of Oct4 was also sufficient to induce pluripotency. Finally, epigenetic manipulation of the Oct4 promoter and enhancer by increasing histone acetylation was sufficient to trigger iPSC induction.

[0091] In the current study, iPSCs were generated by targeting a single gene, Oct4 or Sox2, in the CRISPRa system. Activation of endogenous pluripotency genes had previously been examined in the CRISPRa system, but iPSCs had not been established. Previous studies showed only transient activation of the Oct4 promoter. It is believed that iPSCs were not generated, at least in part, because enhancers were not targeted. In this disclosure, sgRNAs were designed de novo, and sgRNA target sites were selected based on multiple parameters (Figures 5A-5C). The SunTag system used is highly efficient in gene activation and chromatin remodeling, as as many as 24 copies of VP64 could be recruited to the targeting site. This resulted in a 100-fold increase in Oct4 activation, which was much higher than in previous studies (Hu et al., 2014). Moreover, small molecules further enhanced the reprogramming efficiency (Figure 7H). Recently, two studies have shown that VP64 dCas9 VP64 reported the generation of muscle and neuronal cells by activating endogenous MyoD or BAM (Brn2, Ascl1, and Myt1L) in the system (Black et al., 2016; Chakraborty et al., 2014). This disclosure is the first to establish pluripotent stem cells using the CRISPRa method.

[0092] This disclosure mechanistically specifies that direct reconstitution of endogenous Oct4 or Sox2 is sufficient to trigger reprogramming towards pluripotency. This not only provides an alternative method of iPSC generation but also provides insight into the molecular mechanism of pluripotency induction. The Sox2 study demonstrated that endogenous Sox2 activation is a key event in pluripotency induction. It was evident that Sox2 activation was required for S-17 MEF reprogramming, Sox2 reconstitution preceded activation of other key pluripotency genes, and reprogramming efficiency was dependent on Sox2 levels (Figures 3B, 3C, 3G-3I, and 3J). Meanwhile, 20% of the S-17 population activated endogenous Sox2, but only 0.1% could be reprogrammed into EGFP-positive colonies (Figures 3F and 7F), suggesting that the sole activation of endogenous Sox2 is not a determinant of pluripotent cell fate in the context of CRISPRa. Further activation of the Oct4 promoter increased the efficiency of generating EGFP-positive colonies (Figure 3K), supporting the cooperativity of reprogramming multiple pluripotency genes in pluripotency induction. In the Oct4 study, notably, reconstitution of enhancer regions was required for pluripotency induction. Strong transcriptional activation from promoter reconstitution, and modest gene activation from enhancer reconstitution, were observed. However, enhancer reconstitution appears to be essential for further induction of Oct4 at later stages (Figure 4C). This suggests that the Oct4 promoter acts as a fast trigger and that the enhancer is a necessary regulator for latent and higher Oct4 transcription. Whether enhancer reconstitution promoted the establishment of promoter-enhancer loops seen in naive mouse ES cells needs to be further investigated (Kagey et al., 2010). Another interesting point is that this enhancer is one of the 231 super-enhancers specifically found in pluripotent stem cells (Whyte et al., 2013). This disclosure provided functional evidence of a super-enhancer in inducing pluripotency.

[0093] Generation of iPSCs by dCas9-SunTag-p300 core revealed that histone acetylation plays a key role in iPSC generation. Cellular reprogramming involves dynamic epigenetic changes, but whether reprogramming can be achieved through epigenetic manipulation of defined genomic sites was previously unknown. In pluripotent stem cells, histone H3K27 acetylation was highly enriched at both the promoter and enhancer of Oct4, providing an entry to address this issue by manipulating only one type of epigenetic modification. In this disclosure, iPSCs were generated via dCas9-SunTag-p300 core simultaneous targeting of promoters and enhancers. This also paved the way to alter cell fate by site-specific manipulation of epigenetic modifications. Besides p300, several other epigenetic factors (e.g., Tet1, Dnmt3a, KRAB, LSD1) have been identified to function in epigenome editing of activated or silenced genes (Kearns et al., 2015; Liu et al., 2016; Thakore et al., 2015). The expansion of these CRISPR tools raises the possibility of manipulating cell fate by targeting different types of DNA and histone modifications in the future.

[0094] In summary, these data show that the very reconstitution of the endogenous Oct4 or Sox2 loci in the CRISPRa system, here the SunTag system, is sufficient to initiate reprogramming to pluripotency. These studies not only demonstrate the generation of iPSCs by CRISPR activation, but also shed light on the mechanistic understanding of cell reprogramming. As will be further demonstrated in the examples below, the reprogramming strategy is likely to work for the generation of other cell types and in other model systems, such as human cells.

[0095] Example 8. Generation of human iPSCs by CRISPR activation Fibroblast culture and maintenance Human dermal fibroblasts isolated from neonatal foreskin (FTc1007; DFMF030811) were cultured in human fibroblast (hFib) growth medium: Dulbecco's modified Eagle's medium (DMEM) supplemented with 0.1% porcine gelatin (Sigma-Aldrich, St. Louis, MO), 10% fetal bovine serum (FBS), 1% glutagro, 1% non-essential amino acids (NEAA), and 1% penicillin / streptomycin (Corning, Manassas, VA). Routine enzymatic passaging of fibroblasts was performed using Accutase dissociation reagent (Innovative Cell Technologies, Inc., San Diego, CA). Cells were maintained at 37 °C in 5% CO2 and 5% O2.

[0096] Construction of plasmids for expression of human sgRNAs Lentiviral plasmids were constructed using synthetic gBlocks® (Integrated DNA Technologies, Coralville, IA) containing the human U6 promoter, the specific gRNA, and the appropriate homology sequences for NEBuilder® (New England Biolabs Inc., Ipswich, MA) and assembled into the dCas9-Suntag-VP64 plasmid used in the previous examples. The specific gRNA sequences are shown in Table 3. [Table 4]

[0097] Lentiviral packaging HEK293T cells (ATCC CRL-3216) were plated 24 h prior to transfection and transfected with lentiviral packaging plasmids to be 70–80% confluent at the time of transfection. Transfection was performed using Lipofectamine 3000 transfection reagent (Life Technologies, Grand Island, NY). Medium was changed at 16 h. Virus-containing supernatants were collected on days 2 and 3 post-transfection. The supernatants were then passed through a 0.45 μM filter and centrifuged at 20,000 x g for 1.5 h at 4 °C. Concentrated virus was resuspended in DMEM (Corning, Manassas, VA) and aliquots were stored at -80 °C. All viruses were packaged individually and combined when used in two infections. All constructs have been packaged for use with the dCas9-Suntag-VP64 system.

[0098] Virus titration Virus titration was performed using a dilution series on 293T cells seeded the day before. Cells were infected in growth medium, which was DMEM (Corning, Manassas, VA) with 10% FBS (Life Technologies, Grand Island, NY) supplemented with 5 μg / mL polybrene (Millipore, Burlington, MA) and 10 mM HEPES (Corning, Manassas, VA). Cells were centrifuged at 600 x g for 90 min and then incubated for an additional 4–6 h, at which point the transduction medium was replaced with fresh growth medium. Titration was measured by detecting blue fluorescent protein (BFP) expressed from the sgRNA cassette. Samples that showed a positive signal between 1% and 20% for BFP expression were selected for titer calculation using the following formula:

number

[0099] Human fibroblasts were reprogrammed using a CRISPR activation approach involving the exemplary dCas9-Suntag-VP64 system and sgRNA designed to target regulatory DNA elements associated with pluripotency gene transcription and activation of the embryonic genome. All lentiviral components of the Suntag system were packaged separately and delivered through two separate rounds of infection, combining dCas9, VP64, and Tre3G in the first round and sgRNA in the second round, as described in the previous example. Different concentrations of virus were used for comparison. Prior to the second round, transduced cells were treated with 1 μg / mL doxycycline for 24 h to enrich for the first round infected cell population and bulk sorted for expression of GFP-VP64 and dCas9-10XGCN4-P2A-mCherry. For each round of infection, cells were seeded at 5,000 or 10,000 cells / cm. 2 Cells were seeded at a density of 100 x g for 24 h. Twenty-four hours after seeding, transduction was performed with DMEM / F-12 (Corning, Manassas, VA) containing 20% ​​knockout serum replacement (KOSR) (Life Technologies, Grand Island, NY), 1% glutagro, 1% NEAA, 1% penicillin / streptomycin, 10 ng / mL basic human fibroblast growth factor (bFGF) (Life Technologies, Grand Island, NY), and 100 μM β-mercaptoethanol (Life Technologies, Grand Island, NY), 4 μg / mL polybrene (Millipore, Burlington, MA) and 10 mM HEPES (Corning, Manassas, VA). After infection, cells were centrifuged at 600 x g for 90 min and incubated for an additional 4–6 h, at which point the infection medium was replaced with hFib medium containing 1 μg / mL doxycycline to begin induction (day 1). On day 5, cells were plated using Accutase (Innovative Cell Technologies, Inc., San Diego, CA) onto 6-well plates coated with 0.1% porcine gelatin (Sigma-Aldrich, St. Louis, MO) at a density of 7,500 cells / cm.2 Then, doxycycline treatment was discontinued and the medium was changed to one containing a TGFβ receptor inhibitor, a GSK3 inhibitor, a MEK inhibitor, and a ROCK inhibitor. On days 13 and 21, cells were passaged again using Accutase (Innovative Cell Technologies, Inc., San Diego, CA) to a density of 7,500 cells / cm. 2 The cells were seeded on a matrigel (Corning, Manassas, VA) coated 6-well plate at a density of 1000 μg / ml, and the medium was changed to another medium containing GSK3 inhibitor, MEK inhibitor, and ROCK inhibitor but not TGFβ receptor inhibitor.Exemplary TGFβ receptor inhibitors, GSK3 inhibitors, MEK inhibitors, and ROCK inhibitors are known in the art (see, for example, WO2015 / 134652).Specific examples include TGFβ receptor inhibitors SB431542 and A-83-01, MEK inhibitors PD0325901 and PD98059, GSK3 inhibitors CHIR99021 and BIO, ROCK inhibitors thiazovivin and Y27632.

[0100] As early as day 8 and over time, putative iPSCs were observed to have a high nucleus-to-cytoplasm ratio and to display colony morphology similar to ESCs (Figure 9). As shown in Figure 9, inclusion of EEA sgRNA in the CRISPR activation system appears to generate hiPSC clones with stronger morphological features compared to CRISPRa-hiPSC clones obtained without EEA. The observation of the morphology of CRISPRa-hiPSCs generated with or without EEA is consistent with the pluripotency gene expression levels seen under the two conditions (see Figure 10A and B). iPSC colonies generated from the CRISPRa system were further characterized based on the expression of pluripotency markers and multipotent differentiation. Samples were collected for RNA analysis on days 10 and 21 and for flow cytometry on day 21. Cells were fixed for immunofluorescence staining on day 19 and for alkaline phosphatase staining on day 21. Medium changes were performed daily or on other days. Cells were cultured at 37 °C in 5% CO2 and 5% O2. 2中The hiPSCs were cultured at 4°C for 1 h. Phase contrast imaging was performed using an EVOS FL Core Imaging System (Thermofisher, Waltham, MA). Figure 10 shows that expression of endogenous pluripotency genes, such as TRA-1-81, NANOG, SSEA-4 and OCT4, is established in CRISPRA-hiPSC colonies.

[0101] Total RNA was isolated using the RNeasy Plus mini kit (Qiagen, Valencia, CA). Quantitative PCR reactions were performed using the TaqMan RNA-to-CT 1-Step Kit (Applied Biosystems, Foster City, CA) in a reaction volume of 20 μL per well of a 96-well plate and analyzed with a StepOnePlus qPCR system and a QuantStudio 3 qPCR system (Applied Biosystems, Foster City, CA). Reactions were performed in duplicate. Results were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal reference gene. Relative gene expression was quantified using the 2^ΔΔCt method, and data were statistically analyzed using StepOne Software v2.3 and QuantStudio Design and Analysis Software v1.4.1 (Applied Biosystems, Foster City, CA), and GraphPad Prism 7 (GraphPad Software Inc., La Jolla, CA). Primers and probes for Nanog, Oct4, Lin28, Sox2, Rex1, and Gapdh were used for endogenous gene expression assessment. Figure 11 shows that the endogenous pluripotency program is activated over time in the reprogrammed population by activating not only targeted endogenous genes such as Oct4, Sox2, Lin28, and Nanog, but also non-targeted ones, e.g., Rex1. Flow cytometry analysis of TRA-1-81 expression in Figure 12 also demonstrates the established pluripotent state of hiPSCs obtained through CRISPRa at various MOI conditions.

[0102] While the invention has been described in conjunction with the above embodiments, it should be understood that the foregoing descriptions and examples are intended to illustrate, and not to limit, the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

[0103] In addition, when features or aspects of the invention are described in terms of a Markush group, those skilled in the art will recognize that the invention also is described in terms of any individual members or subgroup members of the Markush group.

[0104] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety to the same extent as if each was individually incorporated by reference. In case of conflict, the present specification, including definitions, will control.

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Claims

1. 1. A method for generating induced pluripotent stem cells (iPSCs), comprising: (a) targeting at least one endogenous locus in non-iPSCs using at least one single guide RNA (sgRNA); (b) reconstituting the endogenous locus in the non-iPSCs using a CRISPR activation system and the at least one sgRNA; This results in the generation of iPSCs.

2. (a) the at least one endogenous locus is Oct4, Sox2, Klf4, c-Myc, Nr5a2, Glis1, Cebpa, Lin28, Nanog, or any combination thereof; (b) the at least one endogenous locus comprises a combination of Oct4, Sox2, Klf4, c-Myc, Lin28, and Nanog; (c) the at least one sgRNA targets an Oct4 promoter, an Oct4 enhancer, a Sox2 promoter, a Klf4 promoter, a c-Myc promoter, a Lin28 promoter, a Nanog promoter, an EEA motif, or a combination thereof.

3. (a) the endogenous locus is Oct4; (b) the endogenous locus is Sox2; (c) the sgRNA targeting the Oct4 promoter is selected from SEQ ID NOs: 1-6, 57, and 58; (d) the sgRNA targeting the Oct4 enhancer is selected from SEQ ID NOs: 7-11; (e) the sgRNA targeting the Sox2 promoter is selected from SEQ ID NOs: 12-21 and 59; (f) the sgRNA targeting the Klf4 promoter is selected from SEQ ID NOs: 22-31, and 60; (g) the sgRNA targeting the c-Myc promoter is selected from SEQ ID NOs: 32-41 and 61; (h) the sgRNA targeting the Nr5a2 gene is selected from SEQ ID NOs: 42-45; (i) the sgRNA targeting the Glis1 gene is selected from SEQ ID NOs: 46-50; (j) the sgRNA targeting the Cebpa gene is selected from SEQ ID NOs: 51-56; (k) the sgRNA targeting the Lin28 promoter comprises SEQ ID NO: 62; (l) the sgRNA targeting the Nanog promoter comprises SEQ ID NO: 63; and / or (m) the sgRNA targeting an EEA motif comprises SEQ ID NO:

64.

4. The CRISPR activation system comprises: (a) an inactivated CRISPR-associated nuclease fused to at least one transcriptional activator, and optionally (b) a tandem array of peptides linking an inactivated CRISPR-associated nuclease to the at least one transcriptional activator.

5. (a) the CRISPR activation system comprises an inactivated Cas9 (dCas9); (b) said tandem array of peptides is a SunTag array; (c) the at least one transcriptional activator comprises herpes simplex VP16, a tetramer of VP16 (VP64), or p65.

6. The non-iPSCs include (a) a fibroblast, a skin cell, an umbilical cord blood cell, a peripheral blood cell, or a renal epithelial cell; (b) a mammalian cell, and / or (c) the cell is a human cell, according to claim 1 .

7. (c) contacting the cells of step (b) with a small molecule, including a TGFβR inhibitor, a GSK3 inhibitor, a MEK inhibitor, and a ROCK inhibitor; and optionally, 2. The method of claim 1, further comprising: (d) contacting the generated iPSCs with small molecules, including a GSK3 inhibitor, a MEK inhibitor, and a ROCK inhibitor, but not including a TGFβR inhibitor.

8. 2. The method of claim 1, wherein said reconstitution of said endogenous locus results in a 100-fold or greater level of gene expression compared to without said reconstitution.

9. 1. A method of generating iPSCs, comprising: (a) targeting at least one endogenous locus in non-iPSCs using at least one of an sgRNA targeting a Sox2 gene, an sgRNA targeting an Oct4 promoter, and optionally an sgRNA targeting an Oct4 enhancer or an sgRNA targeting an EEA motif; (b) reconstituting the endogenous locus using a CRISPR activation system and at least one of an sgRNA targeting a Sox2 gene, an sgRNA targeting an Oct4 promoter, and optionally an sgRNA targeting an Oct4 enhancer or an sgRNA targeting an EEA motif; This results in the generation of iPSCs.

10. A composition of a CRISPR activation system, comprising: (a) dCas9; and (b) a SunTag array fused to the dCas9; and (c) at least one acetyltransferase activity domain of p300 (p300 core) bound to the SunTag array.

11. 1. A method of generating iPSCs, comprising: (a) targeting at least one endogenous locus in non-iPSCs using at least one sgRNA; (b) using the CRISPR activation system of claim 10 and the at least one sgRNA to reconstitute the endogenous locus in the non-iPSCs to generate iPSCs.

12. (a) the at least one endogenous locus is Oct4, Sox2, Klf4, c-Myc, Nanog, Lin28, Nr5a2, Glis1, Cebpa, or any combination thereof; (b) the endogenous locus is Oct4; (c) the endogenous locus is Sox2; (d) the at least one sgRNA targets an Oct4 promoter, a Sox2 promoter, or a combination thereof; (e) the at least one sgRNA targets the Oct4 promoter and is selected from SEQ ID NOs: 1-6, 57, and 58; (f) the at least one sgRNA targets the Oct4 enhancer and is selected from SEQ ID NOs: 7-11; (g) the at least one sgRNA targets the Sox2 promoter and is selected from SEQ ID NOs: 12-21, and 59; (h) the at least one sgRNA targets the Klf4 promoter and is optionally selected from SEQ ID NOs: 22-31 and 60; (i) the at least one sgRNA targets a c-Myc promoter and is optionally selected from SEQ ID NOs: 32-41, and 61; (j) the at least one sgRNA targets the Nr5a2 gene, and is optionally selected from SEQ ID NOs: 42-45; (k) the at least one sgRNA targets the Glis1 gene, and is optionally selected from SEQ ID NOs: 46-50; (l) the at least one sgRNA targets the Cebpa gene and is optionally selected from SEQ ID NOs: 51-56; (m) the at least one sgRNA targets the Lin28 promoter and optionally comprises SEQ ID NO: 62; (n) the at least one sgRNA targets the Nanog promoter and optionally comprises SEQ ID NO: 63; 12. The method of claim 11, wherein (o) the at least one sgRNA targets an EEA motif and optionally comprises SEQ ID NO:

64.

13. (a) the non-iPSC is a fibroblast, a skin cell, an umbilical cord blood cell, a peripheral blood cell, or a renal epithelial cell; (b) the non-iPSC is a mammalian cell; and / or 12. The method of claim 11, wherein (c) the non-iPSC is a human cell.

14. 1. A method of generating iPSCs, comprising: (a) targeting at least one endogenous locus in non-iPSCs using at least one of an sgRNA targeting the Oct4 promoter or an sgRNA targeting the Oct4 enhancer; (b) using the CRISPR activation system of claim 23 and at least one of an sgRNA targeting an Oct4 promoter or an sgRNA targeting an Oct4 enhancer to reconstitute the endogenous locus and generate iPSCs.

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