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 on 28 December 2017, the entire disclosure of which is incorporated herein by reference.

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

[0003] Pluripotent stem cells hold considerable promise for regenerative medicine. A better understanding of how endogenous chromatin remodeling leads to pluripotency induction is crucial. Traditionally, 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 initially binds to endogenous loci throughout the genome, remodeling them holistically (Soufi et al., 2012), ultimately leading to the establishment of a pluripotency control circuit.

[0004] However, it is largely unknown which specific rearrangement events in endogenous chromatin trigger reprogramming toward pluripotency. For example, it is unclear whether simultaneous rearrangement of multiple pluripotency-related loci is necessary, or whether the rearrangement of a single locus is sufficient for iPSC induction. In addition, while Oct4, Sox2, and Klf4 target distal elements of many genes necessary for reprogramming (Soufi et al., 2012), how the rearrangement of these distal elements affects pluripotency induction is not well understood. Furthermore, although epigenetic rearrangement is a central mechanism of cellular reprogramming (Smith et al., 2016), it has not been determined whether iPSC induction can be initiated by epigenetic manipulation of any specified endogenous locus. Finally, due to methodological limitations, there is no direct evidence that pluripotency can be induced by the rearrangement of a specific endogenous locus.

[0005] Recently, bacterial-derived type II clustered, regularly spaced short palindromic repeats (CRISPR) and CRISPR-related 9 (Cas9) nuclease systems (CRISPR / Cas9 systems) have once again served their purpose as powerful tools for gene editing in mammalian cells (Cong et al., 2013; Jinek et al., 2012; Mail et al., 2013b). The nuclease-inactivated form of Cas9 (dCas9) has been designed as a programmable synthetic transcription factor when fused with a transactivation domain. This system is called 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 function as a pioneering factor that promotes downstream gene transcription (Polstein et al., 2015). These features make the CRISPRa system a favorable tool for accurately reconstructing endogenous chromatin loci for cell reprogramming from one lineage to another lineage-specific cells (Black et al., 2016; Chakraborty et al., 2014). However, it remains unclear whether this system can generate induced pluripotent stem cells with a wide range of differentiation potential for cells of all lineages, and / or how this system can be made to function. [Overview of the project]

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

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

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

[0009] In other embodiments, the disclosure provides a method for generating iPSCs using a CRISPR activation system comprising dCas9, a SunTag array, and a p300 core.

[0010] In some embodiments, 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 reconstructed. In some embodiments, the single endogenous locus is Oct4 or Sox2.

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

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

[0014] In some embodiments, non-iPSCs are somatic cells, such as fibroblasts, skin cells, umbilical cord blood cells, peripheral blood cells, or renal epithelial cells. In some embodiments, non-iPSCs are mammalian cells. In other embodiments, non-iPSCs are human cells. [Brief explanation of the drawing]

[0015] [Figure 1-1]This study demonstrates the establishment of a pluripotency network in mouse embryonic fibroblasts (MEFs) through gene activation. (A) Scheme showing the dCas9-SunTag-VP64 complex during gene activation. (B) Scheme showing the reprogramming procedure for OG2 MEFs. (C) OG2 MEFs were reprogrammed to form EGFP-positive colonies. Morphology of MEFs on day 0 and reprogrammed colonies on 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 using Dunnett's test. **p<0.01. (E) Colonies showing EGFP signaling 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) Established pluripotency gene expression in CRISPR iPSC and R1 mouse embryonic stem (R1 ES) cells. [Figure 1-2] Same as above. [Figure 2-1]This study demonstrates that Sox2 gene activation is sufficient to generate iPSCs in MEFs. (A) Scheme showing sgRNA targeting sites of the Sox2 promoter, along with the binding peaks of transcription factors (Oct4, Sox2, Nanog), histone acetyltransferase p300, mediator complex, and histone H3K27ac (Whyte et al., 2013). (B) Detection of Sox2 protein by immunofluorescence staining on day 4 (scale bar: 100 μm). (C) Activation of Sox2 in the presence of specified sgRNAs. O-71 and O-127 target the Oct4 promoter, while S-84, S-136, and S-148 target the Sox2 promoter. Data represent mean ± SD (n=4). p-values ​​were determined by unpaired t-tests. **p<0.01. (D) Number of colonies generated by targeting the Oct4 or Sox2 promoter using sgRNA O-71, O-127, S-84, S-136, or S-148. 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 using 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 suitable for germline transmission (K). [Figure 2-2] Same as above. [Figure 3-1]This study demonstrates that rearrangement of the Sox2 promoter triggers reprogramming toward pluripotency in S-17 MEFs. (A) Scheme showing the generation of S-17 MEFs. (B) Sox2 activation over 12 days with and without doxycycline in S-17 MEFs. (C) Histone H3K27 acetylation levels at the Sox2 promoter on 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 an iPSC lineage was established (scale bar: 200 μm). (F) Comparison of the efficiency of SunTag reprogramming and S-17 MEF reprogramming in lentiviruses. (G) Gene expression of Oct4, Nanog, and Rex1 over 12 days in S-17 MEF reprogramming. (H) Histone H3K27 acetylation levels in Oct4, Nanog, and Rex1 promoters on days 0, 4, 8, and 12. (I) Histone H3K27 acetylation levels in Oct4 enhancer on days 0, 4, 8, and 12. (J) Sox2 dependence in S-17 MEF reprogramming. Doxycycline at four different concentrations (0, 0.01, 0.1, and 1 μg / ml) was used to show Sox2 activation (left) and reprogramming efficiency (right). (K) Synergistic effect of Oct4 and Sox2 rearrangement in S-17 MEF reprogramming. Oct4 gene activation (left) and reprogramming efficiency (right) were examined. (L) Total (left) and endogenous Sox2 (right) expression on days 4 and 12 with pluripotency genes overexpressed (OE). Three conditions were tested: Oct4 only, Sox2 only, and OSK (Oct4, Sox2, and Klf4), with mCherry (mCh) overexpression used as a control. (M) Comparison of S-17 MEF reprogramming efficiency and pluripotency gene overexpression. Data in (B), (C), (G), (H), (I), and (K) represent mean ± SD (n=4).The p-value for (B) was determined by a two-way ANOVA using the Bonferroni test, the p-values ​​for (C), (G), (H), and (I) were determined by a one-way ANOVA using Dunnett's test, and the p-value (K) was determined by an independent t-test. **p<0.01; *p<0.05. [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Figure 4-1]To demonstrate the simultaneous reconstruction of the Oct4 promoter and enhancer and reprogram MEFs into iPSCs. (A) Scheme showing sgRNA targeting sites of the Oct4 promoter and enhancer, along with binding peaks and DNase hypersensitivity sites (DHS) of the distribution of transcription factors (Oct4, Sox2, Nanog), histone acetyltransferase p300, mediator complex, and histone H3K27ac (Whyte et al., 2013). (B) Histone H3K27 acetylation levels in the Oct4 enhancer and promoter on day 4. Three different sites upstream of the transcription start site were examined: 2.7kb, 1.4kb, and 0.2kb. (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 simultaneous promoter and enhancer (O-127-2135, O-127-2066, O-127-1965). Four independent experiments are shown. No colonies were observed in the O-127-2135 culture in Experiment 3 and in the O-127-2066 / 1965 culture in Experiment 4. (E) Morphology of EGFP-positive colonies in situ and P0 iPSCs from simultaneous reconstitution of Oct4 promoter and enhancer (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 lines and R1 ES cells. (H) Karyotype analysis of D-9 lines. (I)-(K) In vivo characterization of pluripotent D-9 lines. Chimeric mice were generated using D-9 cells (J), and these cells contributed to gonadal tissue represented by a 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-tests. **p<0.01. [Figure 4-2] Same as above. [Figure 5-1]Shows gene activation by the SunTag system in the differentiation of mouse ES cells and MEFs. (A), (B) Schemes showing the sgRNA targeting sites of the Oct4 promoter and enhancer (A) and the Sox2 promoter (B) together with the binding peaks of the distribution of transcription factors (Oct4, Sox2, Nanog), histone acetyltransferase p300, mediator complex, and histone H3K27ac (Whyte et al., 2013). (C) Genomic DNA sequence (SEQ ID NO: 175) of 500 bp upstream and 100 bp downstream of the Sox2 transcription start site. The binding sites of the sgRNA (blue) and transcription factors (shaded) are highlighted. (D), (E) Transcriptional activation of Oct4, Sox2, Klf4, c-Myc, Nr5a2, Glis1, and Cebpa using sgRNA in the differentiation of mouse ES cells. Schematic diagrams showing the experimental procedure (D) and the fold transcriptional activation using each sgRNA (E). Gal4 sgRNA was used as a negative control. (F), (G) Transcriptional activation of Oct4 and Sox2 using sgRNA in MEFs. Schematic diagrams showing the experimental procedure (F) and the fold transcriptional activation using the selected sgRNAs (G). Gal4 sgRNA was used as a negative control. The sgRNAs targeting the Oct4 promoter (Oct4 Pro) include O-71 and O-127, and the sgRNAs targeting the Oct4 enhancer (Oct4 Enh) include O-1965, O-2066, and O-2135. The sgRNAs targeting the Sox2 promoter (Sox2 Pro) include S-84, S-136, and S-148. (H) Activation of Oct4 and Sox2 using a combination of small molecules, purmorphamine, Chir99021, A83-01, and forskolin (PCAF). The data in (E), (G), and (H) represent the mean ± SD (n = 4). The p-values in (E) and (G) were determined by one-way ANOVA using Dunnett's test, and the p-value in (H) was determined by an unpaired t-test. *p < 0.05; **p < 0.01; ns, not significant. [Figure 5-2] The same as above. [Figure 5-3] The same as above. [Figure 6-1]It is shown 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 from 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) In situ of EGFP-positive colonies and morphology of P0 iPSCs targeting the Oct4, Sox2, Glis1 promoters together (scale bar, 200 μm). (E) Nanog, Sox2, and SSEA-1 staining of EGFP-positive colonies from targeting the Oct4, Sox2, and Glis1 promoters together (scale bar: 200 μm). (F) Number of EGFP-positive colonies generated from targeting the Oct4, Sox2, and Glis1 promoters. Three independent experiments are shown. (G) Examination of the off-target effect of the 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-tests. **p < 0.01; *p < 0.05; ns, not significant. [Figure 6-2] Same as above. [Figure 7-1]This study demonstrates that Sox2 promoter recombination triggers reprogramming toward pluripotency in MEFs. (A) Comparison of Sox2 activation in OG2 and 129MEF with S-84. Data represent mean ± SD (n=4). p-values ​​were determined by unpaired t-tests. **p<0.01. (B) Staining of Sox2 protein in 129MEF on day 4 (scale bar: 200 μm). (C) Generation of reprogrammed colonies in 129MEF (scale bar: 200 μm). (D) Determination of sgRNA cassette copy number in 12 established CRISPR iPSC lines. Clone 8 is the S-17 line as shown. (E) Flow cytometry analysis of BFP in S-17 MEFs. (F) Percentage of Sox2-positive cells on day 4 with and 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) Comparison of coefficients of variation (CV) of reprogramming efficiency of lentivirus SunTag and S-17 MEF. (I) Comparison of reprogramming efficiency with and without PCAF cocktail. (J) Histone H3K27 acetylation levels in the Sox2 promoter on day 4 with pluripotency gene overexpression (OE). Three conditions, Oct4 only, Sox2 only, OSK (Oct4, Sox2, and Klf4) were tested, and mCherry overexpression (mCh OE) was used as a negative control. (K) Comparison of coefficients of variation (CV) of reprogramming efficiency of OSK overexpression and S-17 MEF. (L) S-17 TTF was reprogrammable. The morphology of S-17 TTF on day 0, as well as reprogrammed colonies on days 7 and 14, are shown along with Oct4-EGFP activation (scale bar: 200 μm). [Figure 7-2] Same as above. [Figure 8-1]Simultaneous reconstruction of the Oct4 promoter and enhancer demonstrates the reprogramming of MEFs into iPSCs. (A) Functional verification of a dual sgRNA cassette in differentiating ES cells by introducing two sgRNAs targeting Sox2 (S-148) and c-Myc (M-154). (B) Oct4 protein staining on 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 combined (left) and endogenous (right) Oct4 on day 4 and day 12 with pluripotency genes 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 in the Oct4 enhancer and promoter at day 5 for dCas9-SunTag-VP64 and dCas9-SunTag-p300 core systems. Three different sites upstream of the transcription start site were examined: 2.7kb, 1.4kb, and 0.2kb. (G) Transcriptional activation of Oct4 using dCas9-SunTag-VP64 or dCas9-SunTag-p300 cores over 15 days. (H) Morphology of in-situ EGFP-positive colonies and P1 iPSCs (D-16) generated by manipulating Oct4 promoter and enhancer acetylation using dCas9-SunTag-p300 cores (scale bar, 200 μm). (I) Comparison of pluripotency gene expression in D-16 cell lineage and R1 ES cells. Data for (A), (D), and (G) represent mean ± SD (n=4). The p-values ​​for (A) and (D) were determined by two-way ANOVA using the Bonferroni test, and the p-value for (G) was determined by an independent t-test. **p<0.01; ns, not significant. [Figure 8-2] Same as above. [Figure 9]This demonstrates that CRISPRa produced human iPSC colonies that adopted and maintained pluripotent morphology. The upper panel shows that morphological traces of hiPSCs are more robust when EEA is included in iPSC generation compared to hiPSCs generated without EEA (lower panel at day 28). [Figure 10] This demonstrates the expression of pluripotency genes in human iPSC colonies generated by CRISPRa. A: 28-day hiPSCs generated without EEA and with MOI=1.2. B: 28-day hiPSCs generated with EEA and MOI=1.2. C: 21-day hiPSCs generated with EEA and MOI=6. [Figure 11] We demonstrate that the endogenous pluripotency program is activated over time in reprogrammed populations, as indicated by increased expression of both targeted and non-targeted endogenous genes, including Oct4(Pou5f1), Sox2, Lin28, Nanog, and Rex1. [Figure 12] As demonstrated by flow cytometry, the expression of the pluripotency gene TRA-1-81 increases over time in the reprogrammed population. [Modes for carrying out the invention]

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

[0017] This disclosure is not limited to the specific embodiments described and should therefore be understood to be subject to change. Since the scope of this disclosure is limited only by the appended claims, it should also be understood that the terms used herein are intended solely to describe specific embodiments and are not intended to limit them.

[0018] The detailed description of this disclosure has been divided into various sections for the convenience of the reader, and any disclosure in any section may be combined with that of another section. Any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of this disclosure, but preferred methods and materials are described herein. All publications referenced herein are incorporated by reference to disclose and describe methods and / or materials relating to the reference of those publications.

[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 generally understood by those skilled in the art to which this invention pertains. Terms such as “a,” “an,” and “the” are not intended to refer to a single entity only, but include a general class from which specific examples can be used to illustrate.

[0020] As used herein, the term “generate” means the generation and / or modification of a biological composition (e.g., a cell) from its naturally occurring state by any operation including, but not limited to, altering chromosome structure, nucleotide sequences of polynucleotides (e.g., DNA or RNA), amino acid sequences of polypeptides (e.g., proteins), transcription levels of genes, epigenetic modifications of chromosomal regions, transcription levels of proteins, and transcription levels of RNA.

[0021] As used herein, the terms “reprogramming,” “dedifferentiation,” “increased cellular capacity,” and “increased developmental capacity” refer to methods of increasing the capacity of a cell or dedifferentiating a cell to a less differentiated state. For example, a cell with increased cellular capacity has more developmental plasticity (i.e., can differentiate into more cell types) compared to the same cell that has not been reprogrammed. In other words, a reprogrammed cell is a cell that is less differentiated than the same cell that has not been reprogrammed.

[0022] The terms "pluripotent" or "multipotent" refer to a cell's ability to regenerate and differentiate into multiple tissues or all lineage cell types within the body. For example, embryonic stem cells are a type of multipotent stem cell that can form cells from each of the three germ layers: ectoderm, mesoderm, and endoderm. Pluripotency is a range of developmental capabilities, from incomplete or partial multipotent cells (e.g., epiblast stem cells or EpiSCs) that cannot produce a complete organism to more primitive and multipotent cells that can produce a complete organism (e.g., embryonic stem cells).

[0023] Pluripotency can be partially determined by evaluating the pluripotent properties of cells. Pluripotent features include, but are not limited to, (i) the morphology of pluripotent stem cells; (ii) the potential for unlimited self-renewal; (iii) the 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) the ability to differentiate into all three somatic cell lineages (ectoderm, mesoderm, and endoderm); (v) the formation of teratomas consisting of cells from all three somatic cell lineages; and (vi) the formation of embryoid bodies consisting of cells from all three somatic cell lineages.

[0024] Two types of pluripotency have been described so far: a “priming” or “metastable” state of pluripotency, similar to the epiblastocystem cells (EpiSCs) of a late blastocyst, and a “naive” or “basal” state, similar to the inner cell mass of an early / pre-implantation blastocyst. While both pluripotent states exhibit the characteristics described above, the naive or basal state further exhibits (i) pre-inactivation or reactivation of the X chromosome in female cells, (ii) improved clonality and viability in single-cell culture, (iii) overall reduction in DNA methylation, (iv) reduced deposition of H3K27me3 repressive chromatin marks on developmental regulatory gene promoters, and (v) decreased expression of differentiation markers compared to pluripotent cells in a primed state. 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 possess the characteristics of a pluripotent preparation state. Under standard pluripotent cell culture conditions, such cells remain in a ready state and exhibit basal state characteristics unless exogenous transgene expression is maintained.

[0025] As used herein, “induced pluripotent stem cells” or iPSCs mean that stem cells are generated from differentiated adult, neonatal, or fetal cells that are less differentiated, exhibit pluripotent characteristics, and are artificially reprogrammed to differentiate into any of the three germ layers: endoderm (e.g., lining of the stomach, gastrointestinal tract, lungs), mesoderm (e.g., muscle, bone, blood, genitourinary tract), or ectoderm (e.g., epidermal tissue and nervous system), or dermal tissue. The generated iPSCs do not refer to naturally occurring cells.

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

[0027] As used herein, the term “non-iPSC” refers to any cell that is differentiated or partially differentiated and is not an embryonic stem cell (ESC) or iPSC. Non-iPSCs may originate from any non-germline tissue of the body, including viscera, skin, bone, blood, nerve tissue, and connective tissue.

[0028] As used herein, “CRISPR / Cas system” refers to a bacterial system for defense against foreign nucleic acids. The CRISPR / Cas system is found in a wide range of eubacteria and archaea and includes type I, II, and III subtypes. CRISPR-related nucleases are derived from a family that includes cas, cpf, cse, csy, csn, csd, cst, csh, csa, csm, and cmr. The wild-type type II CRISPR / Cas system utilizes RNA-mediated nucleases in combination with CRISPR RNA (crRNA), and sometimes transactivates crRNA (tracrRNA) to recognize and cleave foreign nucleic acids. For example, in the case of Cas9, when both crRNA and tracrRNA are required, crRNA and tracrRNA can be incorporated into a single guide RNA (sgRNA) molecule that, when combined with Cas9, complementarily discovers and cleaves DNA targets through base pairing between the guide sequence and target DNA sequence within the sgRNA. As used herein, the terms “nuclease-inactivated Cas9” or “dCas9” refer to a modified Cas9 nuclease whose nuclease activity has been neutralized by mutations in the residues of its catalytic RuvC and HNH domains. Neutralizing the catalytic domains converts Cas9 from an RNA-programmable nuclease to an RNA-programmable DNA recognition complex, allowing effectors or markers to be delivered to specific target DNA sequences. Similar operations can be applied to other CRISPR-related nucleases. As used herein, the terms “CRISPR-activating system” or “CRISPRa” refer to a CRISPR / Cas system modified to upregulate gene transcription, where the inactivated Cas nuclease is fused with a transcription activator such as the herpes simplex VP16 transcription activating domain, the VP64 activator (an artificial tetramer of VP16), the p65 activating domain, a synergistic combination of multiple transcription activators, a scaffold peptide that recruits multiple copies of a transcription activator, or a histone acetyltransferase.

[0029] As used herein, the terms “single guide polynucleotide” or “sgPNA” refer to DNA, RNA, or DNA / RNA mixed sequences used in conjunction with the CRISPR / Cas system. The terms “single guide RNA” or “sgRNA” refer to RNA sequences used in conjunction with the CRISPR / Cas system. An sgPNA contains a binding site for a CRISPR-related nuclease, including Cas9, and a guide sequence complementary to the desired target DNA sequence. Base pairing between the sgPNA and the target DNA sequence recruits the CRISPR-related nuclease to the DNA sequence.

[0030] As used herein, the term “target” refers to the process of identifying a nucleic acid sequence (i.e., target) of any composition and / or length at a chromosomal locus, including but not limited to genes, promoters, enhancers, open reading frames, or other chromosomal regions. Next, an sgRNA containing a guide sequence complementary to the pre-identified nucleic acid sequence is designed and synthesized. In some embodiments, the sgRNA directs the CRISPR activation system to the vicinity of the endogenous locus having the pre-identified nucleic acid sequence, based on complementary base pairing of the sgRNA to the pre-identified nucleic acid sequence.

[0031] As used herein, the terms “reconstruct” or “remodel” refer to any modification of gene expression at the transcriptional level. Exemplary gene remodels include, but are not limited to, alterations to the chromatin structure of a gene, supplementation of a gene promoter with a transcription activator, supplementation of a gene enhancer with a transcription activator, and alterations to the histone modifications of a gene by a specific yeast (e.g., histone acetyltransferase).

[0032] II.Cells This invention is based on the discovery that non-iPSCs, such as somatic cells, can be reprogrammed to be pluripotent by activating pluripotency-related genes, either individually or in combination, at endogenous loci through CRISPR activation.

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

[0034] The non-iPSCs of this disclosure may originate from mammals, preferably humans, but include, but are not limited to, non-human primates, murids (i.e., mice and rats), dogs, cats, horses, cattle, sheep, pigs, goats, and the like. In some embodiments, the non-iPSCs are human non-iPSCs.

[0035] III. Targeting and Reconstruction of Endogenous Gene Loci This disclosure relates to targeting and reconstructing endogenous loci using a CRISPR activation system having at least one sgRNA that targets a desired locus. In some embodiments, the endogenous locus is a pluripotency-associated gene. In other embodiments, the endogenous locus is a non-pluripotency-associated gene. Non-limiting examples of pluripotency-associated genes are Oct4, Sox2, Nanog, Klf4, c-Myc, Lin28, Nr5a2, Glis1, Cebpa, Esrrb, and 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 includes using at least one sgRNA in conjunction with a CRISPR activation system targeting a pluripotency-related gene. In some embodiments, the sgRNA targets a pluripotency-related gene selected from the group consisting of Oct4, Sox2, Nanog, Klf4, c-Myc, Lin28, Nr5a2, Glis1, Cebpa, Esrrb, and Rex1.

[0037] In some embodiments, the sgRNA targets the promoter region of a pluripotency-related gene. In some embodiments, the sgRNA targets the enhancer region of a pluripotency-related gene. In some embodiments, the sgRNA targets the promoter region of the Oct4 gene, the enhancer region of the Oct4 gene, the promoter region of the Sox2 gene, or a combination thereof.

[0038] In some embodiments, the sgRNA targets the Oct4 promoter and / or enhancer region located approximately 1 to 5000 bp upstream of the Oct4 transcription start site (TSS), approximately 100 to 4000 bp upstream of the Oct4 TSS, approximately 200 to 3000 bp upstream of the Oct4 TSS, approximately 400 to 4000 bp upstream of the Oct4 TSS, approximately 500 to 3000 bp upstream of the Oct4 TSS, approximately 500 to 3000 bp upstream of the Oct4 TSS, approximately 600 to 2000 bp upstream of the Oct4 TSS, or approximately 700 to 1000 bp upstream of the Oct4 TSS. In some embodiments, the sgRNA is located at or in any region between the Oct4 transcription start site (TSS) at approximately 10-bp, 20-bp, 30-bp, 40-bp, 50-bp, 60-bp, 70-bp, 80-bp, 90-bp, 100-bp, 110-bp, 120-bp, 130-bp, 140-bp, 150-bp, 160-bp, 170-bp, 180-bp, and 1 The sgRNAs target regions of the Oct4 promoter and / or enhancer region upstream of 90-bp, approximately 200-bp, approximately 300-bp, approximately 400-bp, approximately 500-bp, approximately 600-bp, approximately 700-bp, approximately 800-bp, approximately 900-bp, approximately 1000-bp, approximately 1500-bp, approximately 2000-bp, 2500-bp, approximately 3000-bp, 3500-bp, approximately 4000-bp, 4500-bp, and approximately 5000-bp. 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 located approximately 1 to 5000 bp upstream of the Sox2 transcription start site (TSS), approximately 100 to 4000 bp upstream of the Sox2 TSS, approximately 200 to 3000 bp upstream of the Sox2 TSS, approximately 400 to 4000 bp upstream of the Sox2 TSS, approximately 500 to 3000 bp upstream of the Sox2 TSS, approximately 500 to 3000 bp upstream of the Sox2 TSS, approximately 600 to 2000 bp upstream of the Sox2 TSS, or approximately 700 to 1000 bp upstream of the Sox2 TSS. In some embodiments, the sgRNA is located at or between the Sox2 transcription start site (TSS) in any region of approximately 10-bp, 20-bp, 30-bp, 40-bp, 50-bp, 60-bp, 70-bp, 80-bp, 90-bp, 100-bp, 110-bp, 120-bp, 130-bp, 140-bp, 150-bp, 160-bp, 170-bp, and 180-bp. The Sox2 promoter region is targeted at approximately -bp, 190-bp, 200-bp, 300-bp, 400-bp, 500-bp, 600-bp, 700-bp, 800-bp, 900-bp, 1000-bp, 1500-bp, 2000-bp, 2500-bp, 3000-bp, 3500-bp, 4000-bp, 4500-bp, and 5000-bp upstream. In some embodiments, any of the above sgRNAs target the human Sox2 promoter region and / or enhancer region.

[0040] In some embodiments, the sgRNA targets the Nanog promoter and / or enhancer region located approximately 1 to 5000 bp upstream of the Nanog transcription start site (TSS), approximately 100 to 4000 bp upstream of the Nanog TSS, approximately 200 to 3000 bp upstream of the Nanog TSS, approximately 400 to 4000 bp upstream of the Nanog TSS, approximately 500 to 3000 bp upstream of the Nanog TSS, approximately 500 to 3000 bp upstream of the Nanog TSS, approximately 600 to 2000 bp upstream of the Nanog TSS, or approximately 700 to 1000 bp upstream of the Nanog TSS. In some embodiments, the sgRNA is located at or in any region between the Nanog transcription start site (TSS) at approximately 10-bp, 20-bp, 30-bp, 40-bp, 50-bp, 60-bp, 70-bp, 80-bp, 90-bp, 100-bp, 110-bp, 120-bp, 130-bp, 140-bp, 150-bp, 160-bp, 170-bp, 180-bp, and 1 The target regions of the Nanog promoter and / or enhancer region upstream of 90-bp, approximately 200-bp, approximately 300-bp, approximately 400-bp, approximately 500-bp, approximately 600-bp, approximately 700-bp, approximately 800-bp, approximately 900-bp, approximately 1000-bp, approximately 1500-bp, approximately 2000-bp, 2500-bp, approximately 3000-bp, 3500-bp, approximately 4000-bp, 4500-bp, and approximately 5000-bp. In some embodiments, any of the above sgRNAs target the human Nanog promoter region and / or enhancer region.

[0041] In some embodiments, the sgRNA targets the Klf4 promoter and / or enhancer region located approximately 1 to 5000 bp upstream of the Klf4 transcription start site (TSS), approximately 100 to 4000 bp upstream of the Klf4 TSS, approximately 200 to 3000 bp upstream of the Klf4 TSS, approximately 400 to 4000 bp upstream of the Klf4 TSS, approximately 500 to 3000 bp upstream of the Klf4 TSS, approximately 500 to 3000 bp upstream of the Klf4 TSS, approximately 600 to 2000 bp upstream of the Klf4 TSS, or approximately 700 to 1000 bp upstream of the Klf4 TSS. In some embodiments, the sgRNA is located at or in any region between the Klf4 transcription start site (TSS) at approximately 10-bp, 20-bp, 30-bp, 40-bp, 50-bp, 60-bp, 70-bp, 80-bp, 90-bp, 100-bp, 110-bp, 120-bp, 130-bp, 140-bp, 150-bp, 160-bp, 170-bp, 180-bp, and 1 The sgRNAs target regions of the Klf4 promoter and / or enhancer region upstream of 90-bp, approximately 200-bp, approximately 300-bp, approximately 400-bp, approximately 500-bp, approximately 600-bp, approximately 700-bp, approximately 800-bp, approximately 900-bp, approximately 1000-bp, approximately 1500-bp, approximately 2000-bp, 2500-bp, approximately 3000-bp, 3500-bp, approximately 4000-bp, 4500-bp, and approximately 5000-bp. 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 located approximately 1 to 5000 bp upstream of the c-Myc transcription start site (TSS), approximately 100 to 4000 bp upstream of the c-Myc TSS, approximately 200 to 3000 bp upstream of the c-Myc TSS, approximately 400 to 4000 bp upstream of the c-Myc TSS, approximately 500 to 3000 bp upstream of the c-Myc TSS, approximately 500 to 3000 bp upstream of the c-Myc TSS, approximately 600 to 2000 bp upstream of the c-Myc TSS, or approximately 700 to 1000 bp upstream of the c-Myc TSS. In some embodiments, the sgRNA is located at or within the Myc transcription start site (TSS) or any region between it, at approximately 10-bp, 20-bp, 30-bp, 40-bp, 50-bp, 60-bp, 70-bp, 80-bp, 90-bp, 100-bp, 110-bp, 120-bp, 130-bp, 140-bp, 150-bp, 160-bp, 170-bp, 180-bp, and 19-bp. The sgRNAs target the c-Myc promoter and / or enhancer region upstream of 0-bp, approximately 200-bp, approximately 300-bp, approximately 400-bp, approximately 500-bp, approximately 600-bp, approximately 700-bp, approximately 800-bp, approximately 900-bp, approximately 1000-bp, approximately 1500-bp, approximately 2000-bp, 2500-bp, approximately 3000-bp, 3500-bp, approximately 4000-bp, 4500-bp, and approximately 5000-bp. 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 located approximately 1 to 5000 bp upstream of the Nr5a2 transcription start site (TSS), approximately 100 to 4000 bp upstream of the Nr5a2 TSS, approximately 200 to 3000 bp upstream of the Nr5a2 TSS, approximately 400 to 4000 bp upstream of the Nr5a2 TSS, approximately 500 to 3000 bp upstream of the Nr5a2 TSS, approximately 500 to 3000 bp upstream of the Nr5a2 TSS, approximately 600 to 2000 bp upstream of the Nr5a2 TSS, or approximately 700 to 1000 bp upstream of the Nr5a2 TSS. In some embodiments, the sgRNA is located at or in any region between the Nr5a2 transcription start site (TSS) at approximately 10-bp, 20-bp, 30-bp, 40-bp, 50-bp, 60-bp, 70-bp, 80-bp, 90-bp, 100-bp, 110-bp, 120-bp, 130-bp, 140-bp, 150-bp, 160-bp, 170-bp, 180-bp, and 1 The sgRNAs target regions of the Nr5a2 promoter and / or enhancer region upstream of 90-bp, approximately 200-bp, approximately 300-bp, approximately 400-bp, approximately 500-bp, approximately 600-bp, approximately 700-bp, approximately 800-bp, approximately 900-bp, approximately 1000-bp, approximately 1500-bp, approximately 2000-bp, 2500-bp, approximately 3000-bp, 3500-bp, approximately 4000-bp, 4500-bp, and approximately 5000-bp. 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 located approximately 1 to 5000 bp upstream of the Cebpa transcription start site (TSS), approximately 100 to 4000 bp upstream of the Cebpa TSS, approximately 200 to 3000 bp upstream of the Cebpa TSS, approximately 400 to 4000 bp upstream of the Cebpa TSS, approximately 500 to 3000 bp upstream of the Cebpa TSS, approximately 500 to 3000 bp upstream of the Cebpa TSS, approximately 600 to 2000 bp upstream of the Cebpa TSS, or approximately 700 to 1000 bp upstream of the Cebpa TSS. In some embodiments, the sgRNA is located at or in any region between the Cebpa transcription start site (TSS) at approximately 10-bp, 20-bp, 30-bp, 40-bp, 50-bp, 60-bp, 70-bp, 80-bp, 90-bp, 100-bp, 110-bp, 120-bp, 130-bp, 140-bp, 150-bp, 160-bp, 170-bp, 180-bp, and 1 The sgRNAs target regions of the Cebpa promoter and / or enhancer region upstream of 90-bp, approximately 200-bp, approximately 300-bp, approximately 400-bp, approximately 500-bp, approximately 600-bp, approximately 700-bp, approximately 800-bp, approximately 900-bp, approximately 1000-bp, approximately 1500-bp, approximately 2000-bp, 2500-bp, approximately 3000-bp, 3500-bp, approximately 4000-bp, 4500-bp, and approximately 5000-bp. 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 the Esrrb promoter and / or enhancer region located approximately 1 to 5000 bp upstream of the Esrrb transcription start site (TSS), approximately 100 to 4000 bp upstream of the Esrrb TSS, approximately 200 to 3000 bp upstream of the Esrrb TSS, approximately 400 to 4000 bp upstream of the Esrrb TSS, approximately 500 to 3000 bp upstream of the Esrrb TSS, approximately 500 to 3000 bp upstream of the Esrrb TSS, approximately 600 to 2000 bp upstream of the Glis1 TSS, or approximately 700 to 1000 bp upstream of the Esrrb TSS. In some embodiments, the sgRNA is located at or in any region between the Esrrb transcription start site (TSS) at approximately 10-bp, 20-bp, 30-bp, 40-bp, 50-bp, 60-bp, 70-bp, 80-bp, 90-bp, 100-bp, 110-bp, 120-bp, 130-bp, 140-bp, 150-bp, 160-bp, 170-bp, 180-bp, and 1 The sgRNAs target regions of the Esrrb promoter and / or enhancer region upstream of 90-bp, approximately 200-bp, approximately 300-bp, approximately 400-bp, approximately 500-bp, approximately 600-bp, approximately 700-bp, approximately 800-bp, approximately 900-bp, approximately 1000-bp, approximately 1500-bp, approximately 2000-bp, 2500-bp, approximately 3000-bp, 3500-bp, approximately 4000-bp, 4500-bp, and approximately 5000-bp. In some embodiments, any of the above sgRNAs target regions 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 located approximately 1 to 5000 bp upstream of the Rex1 transcription start site (TSS), approximately 100 to 4000 bp upstream of the Rex1 TSS, approximately 200 to 3000 bp upstream of the Rex1 TSS, approximately 400 to 4000 bp upstream of the Rex1 TSS, approximately 500 to 3000 bp upstream of the Rex1 TSS, approximately 500 to 3000 bp upstream of the Rex1 TSS, approximately 600 to 2000 bp upstream of the Rex1 TSS, or approximately 700 to 1000 bp upstream of the Rex1 TSS. In some embodiments, the sgRNA is located at or in any region between the Rex1 transcription start site (TSS) at approximately 10-bp, 20-bp, 30-bp, 40-bp, 50-bp, 60-bp, 70-bp, 80-bp, 90-bp, 100-bp, 110-bp, 120-bp, 130-bp, 140-bp, 150-bp, 160-bp, 170-bp, 180-bp, and 1 The target region is the Rex1 promoter and / or enhancer region upstream of 90-bp, approximately 200-bp, approximately 300-bp, approximately 400-bp, approximately 500-bp, approximately 600-bp, approximately 700-bp, approximately 800-bp, approximately 900-bp, approximately 1000-bp, approximately 1500-bp, approximately 2000-bp, 2500-bp, approximately 3000-bp, 3500-bp, approximately 4000-bp, 4500-bp, and approximately 5000-bp.

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

[0048] IV.CRISPR activation system This disclosure is based on a CRISPR activator derived from the bacterial CRISPR / Cas system. The CRISPR activator includes a type II Cas whose nuclease activity is inactivated and which fuses with an effector to regulate gene transcription. In some embodiments, the dCas is fused with at least one transcription activator. In some embodiments, the dCas is fused with a tandem array of peptides that serve as a scaffold structure for linking 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 the herpes simplex VP16 transcription activator domain (VP64). In one embodiment, the CRISPR activator is dCas-SunTag-VP64, also known as the SunTag CRISPR activator.

[0049] In some embodiments, the CRISPR activator comprises dCas9 fused to a SunTag array and at least one acetyltransferase active domain of p300 (p300 core) bound to the SunTag array. In other embodiments, a method for generating iPSCs comprises modifying histone acetylation at an endogenous locus and thus regulating gene transcription using a CRISPR activator comprising dCas9, a SunTag array, and a p300 core. It is understood that iPSCs can be generated using other CRISPR activators and / or transactivators known to those skilled in the art. [Examples]

[0050] The following examples are intended to further illustrate specific embodiments of the present disclosure. The examples are presented to provide those skilled in the art and are not intended to limit their scope.

[0051] Example 1. Activation of endogenous Oct4 and Sox2 by dCas9-SunTag-VP64 To determine whether and how reconstruction of endogenous loci initiates reprogramming toward pluripotency, the SunTag CRISPR activator was used to precisely reconstruct endogenous pluripotency loci in mouse embryonic fibroblasts (MEFs). dCas9-SunTag-VP64 was selected to enhance its chromatin reconstruction activity by recruiting multiple VP64s to a single targeting site (Figure 1A) (Tanenbaum et al., 2014). dCas9 expression was regulated by the Tet-On promoter. The Oct4 and Sox2 loci were selected as targets due to their central roles in the induction and maintenance of pluripotency.

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

[0053] The sgRNAs were designed to target the Oct4 and Sox2 promoters, as well as the Oct4 enhancer. In addition to the activating effect of the sgRNAs, several factors were considered regarding the targeted genomic sequences, including proximity to but non-overlapping binding sites for pluripotency factors and transcription mechanisms, histone H3K27 acetylation in pluripotent stem cells, and their potential to form promoter-enhancer loops mediated by mediator complexes (Figure 5A-5C).

[0054] For sgRNA constructs, 72-bp oligonucleotides 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 investigated using their respective designed Oct4 and Sox2 sgRNAs 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 for 1 day prior to transfection to reach approximately 70% confluence. VSV-G envelope expression plasmids pMD2.G (Addgene, 12259) and psPAX2 (Addgene, 12260) were used to package the lentiviruses. Plasmids containing the target gene (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. The supernatant containing the virus was collected after 48 hours, filtered through a 0.45 μM filter to remove cell debris, and mixed with 1 volume of fresh medium for immediate use. In the SunTag system, the three lentivirus components (dCas9, VP64, and Tre3G) were individually packaged and mixed at the time of use. SunTag transduction was performed with two rounds of lentivirus infection. The first round was with the SunTag system (dCas9, VP64, and Tre3G), and the second round was with sgRNA. The medium was changed after each infection.

[0056] Mouse ES cells were initially transduced using the dCas9-SunTag-VP64 system and sgRNAs. Since Oct4 and Sox2 are highly expressed in ES cells, the ES cells were differentiated for 4 days in MEF medium supplemented with 1 μM retinoic acid, resulting in decreased Oct4 and Sox2 expression. The dCas9-SunTag-VP64 system, on the other hand, was induced with doxycycline. Analysis of Oct4 expression showed that sgRNAs targeting the narrow promoter region near the transcription start site (TSS) and the 200-bp region of the distal enhancer could increase transcription (Figure 5E). Regarding the Sox2 promoter, sgRNA activity showed a significant tendency towards higher gene activation with sgRNAs closer to the TSS (Figure 5E).

[0057] Selected sgRNAs and dCas9-SunTag-VP64 were also transduced into MEF cells (Figure 5F). MEF cells were incubated with lentiviral supernatant in the presence of 5 μg / ml polybrene (Millipore) for 8 hours or overnight. The promoter was targeted by combining sgRNAs O-127 and O-71, which target the 127- and 71-bp upstream regions of the Oct4 TSS. Similarly, the Oct4 enhancer was targeted by combining O-1965, O-2066, and O-2135, and the Sox2 promoter was targeted by combining S-84, S-136, and S-148 individually. Four days after doxycycline-induced dCas9 induction, targeting the Oct4 promoter resulted in approximately a 100-fold increase in Oct4 transcription, and targeting the enhancer resulted in moderate 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] By detecting blue fluorescent protein (BFP) from sgRNA cassettes, viral titer can be determined in primary MEF cells, and the multiplicity of infection (MOI) can be calculated using the Poisson distribution (Arai et al., 1999). P(k)=e -m m k / k! Here, 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 the 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 was as follows: [Table 1]

[0059] Example 2. Establishment of the MEF pluripotency network by gene activation using dCas9-SunTag-VP64. Next, we 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 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 to 10 sgRNAs were designed and tested in differentiating ES cells (Figure 5E). 1 to 3 sgRNAs for each promoter were included in the previous Oct4 / Sox2 sgRNA pool (see Table 1). Next, a small molecule cocktail consisting of Parnath, Chir99021, A83-01, and forskolin (PCAF) was added to the reprogramming medium. This chemical cocktail further increased Oct4 and Sox2 transcription by 3-4 times on 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 exhibit a strong EGFP signal when endogenous Oct4 is actively transcribed (Szabo et al., 2002). MEF cells were introduced at a rate of 10,000 cells / cm³ 24 hours prior to transduction. 2 Cells were seeded at a density onto gelatin-coated plates. After transduction with dCas9-SunTag-VP64 and an sgRNA pool (18 sgRNAs in total, see Table 1), the cells were recovered in MEF medium for 24 hours. To initiate reprogramming, the MEF medium was changed to reprogramming medium supplemented with 1 μg / ml doxycycline (ES medium supplemented with 10 μM Parnath, 3 μM Chir99021, 1 μM A83-01, and 10 μM forskolin). This was called day 0 (Figure 1B). On day 3, cells were treated with 1 mg / ml collagenase B (Roche) for 20 minutes, followed by treatment with 0.05% trypsin for 5 minutes at 37°C, and then refilled into new wells (30,000 cells / cm²). 2 The cells were re-seed and 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 observed (Figure 1C). These colonies were also positive for Nanog, Sox2, and SSEA-1 (Figure 1F). Throughout the entire process, the medium was changed every other day for the first 12 days. Thereafter, standard ES medium was used and changed daily, and the EGFP-positive colonies were typically ready for iPSC induction between 16 and 18 days.

[0061] Next, EGFP-positive colonies were expanded on feeder cells to create CRISPR iPSC lines. For iPSC induction, reprogrammed cultures were incubated with 1 mg / ml collagenase B (Roche) at 37°C for 20 minutes. Single colonies were picked under a microscope and digested with 0.05% trypsin for 5–10 minutes to obtain single-cell suspensions. These cells were then seeded into feeders in standard ES medium, and these cells were considered P0 iPSCs. The CRISPR iPSCs formed dome-shaped colonies similar to typical mouse ES cells with a strong EGFP signal (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 loss of EGFP signal or ES morphology (Figure 1E). These data demonstrate established pluripotency in these CRISPR iPSCs.

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

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

[0064] Next, it was determined whether targeting the Oct4, Sox2, and Glis1 promoters together was sufficient to generate iPSCs. Since 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 gene (Figure 6B), one sgRNA was selected to target the promoter of each gene: O-127 for Oct4, S-84 for Sox2, and G-215 for Glis1. This approach simplifies the reprogramming system and may 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 strains could be established (Figures 6D and 6E).

[0065] During OSG reprogramming, it was surprisingly observed 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 rule out any possible off-target effects of S-84, the top 10 predicted targets for S-84 were examined. Off-target sgRNAs were predicted by the 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). The Sox2 protein was also detected on day 4 (Figure 2B). Furthermore, the reprogramming procedure was repeated using two other 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] Next, the reliability of the pluripotency of these iPSCs was investigated. Nanog and SSEA-1 proteins were also detected within these EGFP-positive colonies, establishing a CRISPR iPSC line (Figures 2E and 2F). For line S-17, the expression of major pluripotency factors was similar to that in R1 mouse ES (R1 ES) cells (Figure 2G). Karyotyping of the iPSC lines was performed using Cell Line Genetics, and Giemsa junctions were analyzed. The generated iPSCs were also karyotypically normal (Figure 2H).

[0067] S-17 cells are B6 albino (B6(Cg)-Tyr c-2J (J, Jackson Labs) More rigorous assays for pluripotency were performed by injecting blastocysts into a background. EGFP-positive cells were found in the gonadal regions of 71.4% (5 out of 7) of E13.5 embryos (Figure 2I). Production 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 tests, and S-17 cells were also suitable for germline (Figure 2K). These data led to the conclusion that single-gene coordinate formation of the Sox2 promoter by a single sgRNA was sufficient to reprogram MEFs into true pluripotent stem cells.

[0068] R1 ES cells were maintained in feeders containing 5% ES-FBS (Invitrogen) and 15% KO-serum substitute (Invitrogen), 1% GlutaMAX™ (Invitrogen), 1% non-essential amino acids (Invitrogen), 55 μM 2-mercaptoethanol (Sigma), 10 ng / ml leukemia suppressor (Stemgent), 3 μM CHIR99021, and 1 μM PD0325901 in KO-DMEM (Invitrogen). In 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 suppressor, 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] In the generation of chimeric mice and germ cell transmission tests, B6(Cg)-Tyr was produced from superovulated 4-week-old mice. c-2J / J(B6-albino) mice (Jackson Laboratory) were prepared for blastocyst development using B6(Cg)-Tyr c-2J The mice were mated with male J mice. Morulas (2.5 days post-coital) were collected and cultured overnight in KSOM medium (Millipore) at 37°C and 5% CO2. The following morning, the blastocysts were ready for iPSC injection, and approximately 10-20 cells were injected into each blastocyst. The injected blastocysts were cultured in KSOM medium at 37°C and 5% CO2 for 1-2 hours and then transplanted into the uterus of pseudo-pregnant CD1 female mice (Charles River, stock #022) 2.5 days post-coital. Chimeric mice can be identified by the color of their mosaic coat. Male chimeric mice were further converted into female B6(Cg)-Tyr c-2J / J mice were crossed with offspring mice exhibiting black coat coloration, which were considered to have successfully transmitted germline impurities. Regarding gonadal contribution, the injected embryos recovered at 10 days post-transplant (E13.5). The gonadal region of each embryo was collected and EGFP signaling was visualized under a microscope. All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of California, San Francisco.

[0070] Example 4. S-17 MEF is reprogrammable, highly efficient, and has low variability. Lentiviral transduction-induced reprogramming efficiency was relatively low and varied in both OG2 and 129 mice (129S2 / SvPasCrl, Charles River), resulting in MEF (0-0.013%) (Figure 2C, 6A, 6F, 7A-7C). This may be due to inefficient delivery of SunTag components and the random number of copies of components delivered to cells. This is reflected in the varying copy numbers of sgRNA cassettes in the genomes of established iPSC lines, with 1-5 copies found per cell line out of 12 lines (Figure 7D). For quantification of sgRNA cassettes 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). Sox2 amplification was used to normalize the genomes of each cell line. Plasmids containing the target were used to construct standard curves. Calibration curves were constructed by plotting Ct values ​​against plasmid copy numbers for a series of plasmid dilutions (10 pg, 1 pg, 0.1 pg, 0.01 pg) and then plotting the copy numbers of approximately 30 ng of Sox2 gene and sgRNA cassette. Genomic DNA was calculated based on a standard curve. Next, sgRNA copy numbers were calculated by normalizing to Sox2 gene (2 copies / cell). [Table 3-1] [Table 3-2] [Table 3-3]

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

[0072] Next, we investigated whether S-17 MEFs were reprogrammable. Sox2 transcription was significantly upregulated on 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 transcriptions were detected on day 8 and subsequently increased dramatically (Figure 3G). Meanwhile, morphological changes were observed from day 4, and EGFP-positive colonies appeared on day 7 (Figure 3E). iPSC lines were also established (Figure 3E). Using S-17 MEFs, the 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 specified time using the RNeasy Plus Mini Kit containing QiaShredder (Qiagen) and treated with the DNA-free Kit (Ambion) to remove genomic DNA. RNA was reverse transcribed using the iScript cDNA Synthesis Kit (Bio-Rad). Quantitative PCR was performed using iQTM SYBR Green Supermix (Bio-Rad) on a 7500 Fast Real-Time PCR System (Applied Biosystems). All reactions were performed in quadruplicate. All data were statistically analyzed with Prism7. For immunofluorescence staining, cells were washed three times with DPBS and fixed with 4% PFA at 4 °C for 30 minutes. Blocking was performed using donkey serum (10% in DPBS) at 4 °C for 1 hour. Antibodies were diluted with 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] It was also tested whether more differentiated tail tip fibroblasts (TTF) were reprogrammable. S-17 TTF was derived from 14-month-old adult chimeric mice. The tails were removed, minced into 1 mm pieces, and cultured in 6 cm dishes. The medium was changed half every 3 days until fibroblasts migrated out of the explants. Induced TTF was maintained in DMEM supplemented with 10% FBS and non-essential amino acids and was ready to use (P1) immediately after subculture. In the presence of doxycycline, TTF underwent morphological changes and EGFP-positive colonies were obtained within 2 weeks (Figure 7L). These observations indicate that both S-17 MEF and TTF are reprogrammable.

[0075] For reprogramming by S-17 MEF or mouse TTF, MEF or TTF was seeded at 5,000 cells / cm 2Seeds were seeded at the specified density. After 24 hours, the medium was switched to reprogramming medium containing 1 μg / ml doxycycline. This was shown as day 0. The medium was changed every other day until day 14. EGFP-positive colonies were counted for calculations of reprogramming efficiency or used for the derivation of iPSC lines.

[0076] Example 5. Reconstruction of the Sox2 promoter triggers reprogramming toward pluripotency of the S-17 MEF. Without doxycycline, Sox2 activation could not be detected, and no colonies were obtained (Figures 3B and 3J). Removal of the PCAF cocktail from the medium resulted in the generation of EGFP-positive colonies, although efficiency was reduced (Figure 7I). This observation led to the conclusion that endogenous Sox2 activation triggers S-17 MEF reprogramming.

[0077] Next, we investigated 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 showed a positive correlation with doxycycline concentration, and reprogramming efficiency was clearly dependent on Sox2 levels (Figure 3J).

[0078] Since VP64 promotes gene transcription and chromatin rearrangement by employing multiple epigenetic modifiers (Hirai et al., 2010), we investigated whether and how the SunTag system can epigenetically rearrange the Sox2 promoter. Chromatin immunoprecipitation (ChIP) was performed using an H3K27 acetylated (H3K27ac) antibody against the Sox2 promoter. All ChIP experiments were performed using the EZ-ChIP chromatin immunoprecipitation kit (Millipore, 17-371), following the kit's protocol with modifications. In short, approximately 2 × 10⁻⁶ 6Cells were crosslinked in 10 ml of growth medium with 0.275 ml of 37% formaldehyde. 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. Next, genomic DNA was sheared to lengths of 100–500 bp using a Covaris S2 sonicator under optimized conditions. 1.5 μg of H3K27 acetylated antibody (Abcam, ab4729) and 15 μL of magnetic protein A / G beads (Millipore 16-663) were used for each sample. Finally, the DNA fragments were eluted with 50 μl of elution buffer C and used for downstream qPCR.

[0079] As early as day 4, H3K27ac levels had already doubled, and increased further on days 8 and 12 (Figure 3C). This indicates that the SunTag system targeting Sox2 induced a stepwise and consistent epigenetic rearrangement in the Sox2 promoter. Epigenetic rearrangements of the Oct4, Nanog, and Rex1 promoters were also tested, and their H3K27ac levels increased significantly with a 4-day latency, similar to gene transcription (Figures 3G and 3H). Interestingly, the Oct4 enhancer showed a simultaneous increase in H3K27ac levels (Figure 3I). These data suggest that Sox2 activation facilitated the subsequent induction of other key genes for pluripotency establishment.

[0080] Next, we tested whether additional targeting of the Oct4 promoter in S-17 MEFs enhanced reprogramming efficiency. Transduction of O-127 resulted in a significant increase in Oct4 transcription and also increased reprogramming efficiency (Figure 3K). This synergistic effect supported the idea that Oct4 and Sox2 cooperate in inducing pluripotency.

[0081] S-17 MEF reprogramming was compared with conventional reprogramming using overexpressed factors. Unlike S-17 MEF, the overexpressed factors failed to epigenetically reconstruct the Sox2 promoter on day 4 (Figure 7J), and Sox2 transcription at the endogenous locus was not effectively detected on 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 MEF, with greater inter-experimental variability (Figures 3M and 7K).

[0082] Example 6. Simultaneous reconstruction of the Oct4 promoter and enhancer reprograms the MEF to iPSC. Reconstruction of both the Oct4 promoter and enhancer is crucial for pluripotency induction, and targeting the promoter alone was insufficient for generating EGFP-positive colonies (Figures 6A and 2C). Given that major pluripotency factors, as well as the p300 and mediator complex, are enriched in the Oct4 distal enhancer of mouse ES cells (Figure 4A), it was hypothesized that simultaneous reconstruction of the Oct4 promoter and enhancer is necessary for pluripotency induction.

[0083] To test the hypothesis, a dual sgRNA cassette transcribed 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 using a single sgRNA construct as a template, with primers mU6-T2H-F (SEQ ID NO: 167 ctaggatccattaggcGGGTACAGTGCAGGGGAA) and mU6-T2H-R2 (SEQ ID NO: 168 atacggttatccacgcGGCCGCCTAATGGATCCT). This fragment was purified and used in a recombination reaction with another construct containing the first sgRNA digested with NotI. The second mU6-sgRNA cassette is downstream of the first cassette in the same transcription direction.

[0084] O-127-2066 simultaneously reconstituted the Oct4 promoter and enhancer with elevated levels of H3K27ac (Figure 4B). Gene transcription levels in O-127-2066 were similar to those in O-127 on days 4 and 8 (Figure 4C). However, Oct4 transcription increased further in O-127-2066 cultures after day 8. In particular, overall Oct4 expression in the population increased dramatically when cells were reseeded on days 7 and 11 to allow cell proliferation (Figure 4C). In both O-127 and O-2066 cultures, weak Oct4 expression remained largely unchanged after 8 days (Figure 4C). Thus, by day 12, EGFP-positive colonies were observed in O-127-2066 cultures, and these colonies also expressed Nanog, Sox2, and SSEA-1, demonstrating an acquired core pluripotency network (Figures 4E and 4F). iPSC lines could be induced from these colonies (Figure 4E). On the other hand, no colonies were observed in O-127 or O-2066 cultures (Figure 4D).

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

[0086] A truly pluripotent stem cell lineage was also achieved. The D-9 lineage exhibited pluripotency gene expression and a normal karyotype similar to R1 ES cells (Figures 4G and 4H). After injecting D-9 cells into B-6 albino blastocysts, 60% of the offspring produced living chimeric mice (6 out of 10) (Figure 4J). This lineage significantly contributed 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 mice (2 out of 4) (Figure 4K).

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

[0088] For the cloning of the p300 core, the backbone was digested from pSLQ1711-pPGK-ScFV(GCN4)-sfGFP-VP64 with SbfI-HF and RsrII(NEB) and obtained using a gel purification kit (Qiagen). An 83-bp SV40 nuclear localization site (NLS) with a linker was cloned and added between sfGFP and the 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 the 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 using the dCas9-SunTag-p300 core system. The p300 core cultures showed comparable H3K27ac levels to VP64 with the Oct4 promoter and enhancer, but only 1 / 30th of Oct4 transcription was detected in the p300 core cultures at day 5 (Figures 8F and 8G). This can be explained by the p300 core's inability to employ the transcriptional mechanism. The cultures were then subculturified at days 9 and 14. Interestingly, by day 10, Oct4 levels were comparable between the VP64 and p300 core cultures (Figure 8G). Consequently, EGFP-positive colonies were generated in the p300 core cultures, and iPSC lines were produced (Figures 8H and 8I). These observations indicate that the manipulation of histone acetylation by the p300 core led to chromatin remodeling similar to that of VP64, but with a significant latency for transcriptional activation. Together, these results indicate that epigenetic reconstruction of Oct4 promoters and enhancers via VP64 or p300 cores is sufficient to trigger reprogramming toward pluripotency.

[0090] Due to the widespread binding of ectopic proteins, identifying the reconfiguration events on endogenous chromatin that trigger reprogramming toward pluripotency is extremely challenging. To gain insight into this, we precisely reconfigured specific sites on endogenous chromatin using the CRISPRa system, dCas9-SugTag-VP64, or dCas9-SunTag-p300 cores. For the first time, we discovered that targeted reconfiguration of either Oct4 or Sox2 single genes was sufficient to initiate reprogramming toward pluripotency, establishing a true pluripotent stem cell line. S-17 MEFs and TTFs derived from CRISPR iPSCs were also reprogrammable, suggesting that activation of endogenous Sox2 is a key event in pluripotency induction. Simultaneous reconfiguration of the Oct4 promoter and enhancer was also sufficient to induce pluripotency. Finally, epigenetic manipulation of the Oct4 promoter and enhancer by increasing histone acetylation was sufficient to induce iPSC induction.

[0091] In current research, iPSCs were generated by targeting a single gene, either Oct4 or Sox2, using the CRISPRa system. While activation of endogenous pluripotency genes had been previously examined using the CRISPRa system, iPSC generation had not been established. Previous studies had only shown transient activation of the Oct4 promoter. It is thought that iPSCs were not generated, at least partially, because the enhancer was not targeted. In this disclosure, a new sgRNA was designed, and the sgRNA target site was selected based on multiple parameters (Figures 5A-5C). The SunTag system used is highly efficient in gene activation and chromatin rearrangement because up to 24 copies of VP64 can be employed at the target site. As a result, Oct4 activation increased 100-fold, which was significantly higher than in previous studies (Hu et al., 2014). Furthermore, the small molecule further enhanced the reprogramming efficiency (Figure 7H). Recently, two studies have shown that VP64 dCas9 VP64 We have 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 rearrangement of endogenous Oct4 or Sox2 is sufficient to trigger reprogramming toward pluripotency. This not only provides an alternative method for iPSC generation but also offers insights into the molecular mechanisms of pluripotency induction. Sox2 studies have demonstrated that activation of endogenous Sox2 is a critical event in pluripotency induction. It was revealed that Sox2 activation is required for S-17 MEF reprogramming, Sox2 rearrangement precedes activation of other major pluripotency genes, and the efficiency of reprogramming was dependent on Sox2 levels (Figures 3B, 3C, 3G-3I, and 3J). On the other hand, while 20% of the S-17 population activated endogenous Sox2, only 0.1% were able to reprogram into EGFP-positive colonies (Figures 3F and 7F), suggesting that 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 EGFP-positive colony generation (Figure 3K), supporting the cooperativeness of reprogramming of multiple pluripotency genes in pluripotency induction. In Oct4 studies, in particular, enhancer region reconstruction was required for pluripotency induction. Strong transcriptional activation from promoter reconstruction and moderate gene activation from enhancer reconstruction were observed. However, enhancer reconstruction appears to be essential for further induction of Oct4 at a later stage (Figure 4C). This suggests that the Oct4 promoter acts as a fast trigger and the enhancer is a regulator necessary for potential and higher Oct4 transcription. Further investigation is needed to determine whether enhancer reconstruction facilitated the establishment of the promoter-enhancer loop observed in naive mouse ES cells (Kagey et al., 2010). Another interesting point is that this enhancer is one of the 231 superenhancers particularly found in pluripotent stem cells (Whyte et al., 2013). This disclosure provided functional evidence for super-enhancers in pluripotency induction.

[0093] The generation of iPSCs using the dCas9-SunTag-p300 core revealed that histone acetylation plays a crucial role in iPSC generation. While cellular reprogramming involves dynamic epigenetic changes, it was previously unknown whether reprogramming could be achieved through epigenetic manipulation of defined genomic sites. In pluripotent stem cells, histone H3K27 acetylation is highly enriched in both the promoter and enhancer of Oct4, providing an entry point to address this problem by manipulating only one type of epigenetic modification. In this disclosure, iPSCs were generated via simultaneous targeting of the dCas9-SunTag-p300 core in both the promoter and enhancer. This also paved the way for altering cell fate through site-specific manipulation of epigenetic modifications. In addition to p300, several other epigenetic factors (such as Tet1, Dnmt3a, KRAB, and LSD1) have been identified as functioning in the epigenome editing of activation or silencing genes (Kearns et al., 2015; Liu et al., 2016; Thakore et al., 2015). The expansion of these CRISPR tools will increase the potential to manipulate cell fate by targeting a wider variety of DNA and histone modifications in the future.

[0094] In summary, these data demonstrate that the very reconstruction of the CRISPRa system, in this case the SunTag system, the endogenous Oct4 or Sox2 locus is sufficient to initiate reprogramming to pluripotency. These studies not only demonstrate iPSC generation by CRISPR activation but also shed light on the mechanistic understanding of cellular reprogramming. As further demonstrated in the following examples, the reprogramming strategy is thought to function in 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 Medium (DMEM) with 0.1% porcine gelatin (Sigma-Aldrich, St. Louis, MO). They were treated with 10% fetal bovine serum (FBS), 1% glutaglobulin, 1% non-essential amino acids (NEAA), and 1% penicillin / streptomycin (Corning, Manassas, VA). Standard enzymatic passage 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] A lentiviral plasmid for human sgRNA expression was constructed using synthetic gBlocks® (Integrated DNA Technologies, Coralville, IA) containing a human U6 promoter, a specific gRNA, and a suitable homologous sequence for NEBuilder® (New England Biolabs Inc., Ipswich, MA), and assembled into the dCas9-Suntag-VP64 plasmid used in previous examples. The specific gRNA sequence is shown in Table 3. [Table 4]

[0097] Lentivirus-packaged HEK293T cells (ATCC CRL-3216) were plated 24 hours prior to transfection and transfected with lentivirus packaging plasmids to achieve 70-80% confluence at transfection. Transfection was performed using Lipofectamine 3000 transfection reagent (Life Technologies, Grand Island, NY). The medium was changed every 16 hours. The supernatant containing the virus was collected on days 2 and 3 post-transfection. The supernatant was then filtered through a 0.45 μM filter and centrifuged at 20,000 xg for 1.5 hours at 4°C. The concentrated virus was resuspended in DMEM (Corning, Manassas, VA), and aliquots were stored at -80°C. All viruses were individually packaged and combined when used for two infections. All constructs are packaged for use in the dCas9-Suntag-VP64 system.

[0098] Viral titration was performed using a series of dilutions on 293T cells seeded the previous day. Cells were infected in growth medium, which was DMEM (Corning, Manassas, VA) containing 10% FBS (Life Technologies, Grand Island, NY), supplemented with 5 μg / mL polyblen (Millipore, Burlington, MA) and 10 mM HEPES (Corning, Manassas, VA). Cells were centrifuged at 600xg for 90 minutes, followed by incubation for a further 4–6 hours, at which point the transduction medium was replaced with fresh growth medium. Titration was measured by detecting blue fluorescent protein (BFP) expressed from sgRNA cassettes. Samples showing a positive signal of 1% to 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 an exemplary dCas9-Suntag-VP64 system and sgRNA, designed to target regulatory DNA elements associated with pluripotency gene transcription and embryonic genome activation. All lentiviral components of the Suntag system were packaged individually and introduced through two separate infection rounds, combining dCas9, VP64, and Tre3G in the first round and sgRNA in the second round, as described in the previous example. Various concentrations of virus were used for comparison. Prior to the second round, transduced cells were treated with 1 μg / mL doxycycline for 24 hours to enrich the first-infected cell population and bulk-sorted for expression of GFP-VP64 and dCas9-10XGCN4-P2A-mCherry. For each round of infection, cells were expressed at 5,000 or 10,000 cells / cm². 2 Seeds were seeded at the following density. 24 hours after seeding, transduction was performed with DMEM / F-12 (Corning, Manassas, VA) containing 20% ​​knockout serum substitution (KOSR) (Life Technologies, Grand Island, NY), 1% glutaglobulin, 1% NEAA, 1% penicillin / streptomycin, 10 ng / mL basic human fibroblast growth factor (bFGF) (Life Technologies, Grand Island, NY), 100 μM β-mercaptoethanol (Life Technologies, Grand Island, NY), 4 μg / mL polyblen (Millipore, Burlington, MA), and 10 mM HEPES (Corning, Manassas, VA). After infection, cells were centrifuged at 600xg for 90 minutes and incubated for a further 4-6 hours. At this point, the infection medium was replaced with hFib medium containing 1 μg / mL doxycycline to initiate induction (Day 1). On day 5, cells were loaded at a density of 7,500 cells / cm³ into 6-well plates coated with 0.1% porcine gelatin (Sigma-Aldrich, St. Louis, MO) using Accutase (Innovative Cell Technologies, Inc., San Diego, CA).2 Doxycycline treatment was discontinued, and the culture 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, the cells were passaged again using Accutase (Innovative Cell Technologies, Inc., San Diego, CA) to 7,500 cells / cm³. 2 Seeds were seeded at a density in Matrigel (Corning, Manassas, VA) coated 6-well plates, and the medium was changed to a different medium containing a GSK3 inhibitor, a MEK inhibitor, and a ROCK inhibitor, but without a 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 the TGFβ receptor inhibitors SB431542 and A-83-01, the MEK inhibitors PD0325901 and PD98059, the GSK3 inhibitors CHIR99021 and BIO, and the ROCK inhibitors thiazovibin and Y27632.

[0100] As early as day 8 and over time, iPSCs were observed that exhibited a high nucleus-to-cytoplasm ratio and colony morphology similar to ESCs (Figure 9). As shown in Figure 9, including EEA sgRNA in the CRISPR activating system appears to generate hiPSC clones with stronger morphological features compared to CRISPRa-hiPSC clones obtained without EEA. The morphological observations of CRISPRa-hiPSCs generated with and without EEA are consistent with the pluripotency gene expression levels observed under the two conditions (see Figures 10A and B). iPSC colonies generated from the CRISPRa system were further characterized based on the expression of pluripotency markers and multipotency differentiation. Samples were collected for RNA analysis on day 10 and day 21 and 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 fixed at 37°C in 5% CO2 and 5% O 2中The cells were cultured in [specimen type]. Phase-contrast imaging was performed using the EVOS FL core imaging system (Thermofisher, Waltham, MA). Figure 10 shows that the 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 was performed using the TaqMan RNA-to-CT 1-step kit (Applied Biosystems, Foster City, CA) at a reaction volume of 20 μL per well in a 96-well plate, and the results were analyzed using the StepOnePlus qPCR system and QuantStudio 3 (Applied Biosystems, Foster City, CA). The reaction was performed twice. Results were normalized against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the internal reference gene. Relative gene expression was quantified using the 2^ΔΔCt method, and the 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 to evaluate endogenous gene expression. 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 untargeted ones such as Rex1. Flow cytometry analysis of TRA-1-81 expression in Figure 12 also demonstrates the established pluripotency state of hiPSCs obtained through CRISPRa under various MOI conditions.

[0102] While the present invention has been described in relation to the embodiments described above, it should be understood that the foregoing description and examples are intended to illustrate, and not 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 whom the invention relates.

[0103] In addition, if any feature or aspect of the present invention is described in relation to the Markush group, a person skilled in the art will recognize that the present invention may also be described in relation to any individual member or subgroup member of the Markush group.

[0104] All publications, patent applications, patents, and other references mentioned herein are explicitly incorporated by reference to the same extent as each one is incorporated by reference individually. In case of any conflict, this specification, including definitions, shall prevail.

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Claims

1. A composition for generating induced pluripotent stem cells (iPSCs) from non-induced pluripotent stem cells, At least two single guide RNAs (sgRNAs) targeting at least one endogenous locus of a non-iPSC, or polynucleotides encoding said at least two single guide RNAs (sgRNAs), The CRISPR activation system for reconstructing the at least one endogenous gene locus or a polynucleotide encoding the CRISPR activation system, A composition wherein the at least one endogenous locus comprises Oct4, and when introduced into the non-iPSC, the first of the at least two sgRNAs targets the Oct4 promoter, and the second of the at least two sgRNAs targets the Oct4 enhancer.

2. The composition according to claim 1, comprising a polynucleotide encoding at least two sgRNAs and a polynucleotide encoding the CRISPR activation system.

3. The composition according to claim 1, comprising the at least two sgRNAs and the CRISPR activation system.

4. (a) The at least one endogenous locus further comprises 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 endogenous gene locus further comprises Sox2 and / or (d) The composition according to claim 1 or 2, wherein the at least two sgRNAs further target the Sox2 promoter, the Klf4 promoter, the c-Myc promoter, the Lin28 promoter, the Nanog promoter, the EEA motif, or a combination thereof.

5. (a) The sgRNA targeting the Oct4 promoter includes an RNA sequence corresponding to any of SEQ ID NOs: 1-6, 57, and 58. (b) The sgRNA targeting the Oct4 enhancer comprises an RNA sequence corresponding to any of SEQ ID NOs: 7 to 11, (c) The sgRNA targeting the Sox2 promoter comprises an RNA sequence corresponding to any of SEQ ID NOs. 12-21 and 59, (d) The sgRNA targeting the Klf4 promoter comprises an RNA sequence corresponding to any of SEQ ID NOs. 22-31 and 60, (e) The sgRNA targeting the c-Myc promoter comprises an RNA sequence corresponding to any of SEQ ID NOs. 32-41 and 61, (f) The sgRNA targeting the Nr5a2 gene includes an RNA sequence corresponding to any of sequence numbers 42 to 45, (g) The sgRNA targeting the Glis1 gene includes an RNA sequence corresponding to any of sequence numbers 46 to 50, (h) The sgRNA targeting the Cebpa gene includes an RNA sequence corresponding to any of sequence numbers 51 to 56, (i) The sgRNA targeting the Lin28 promoter includes the RNA sequence corresponding to SEQ ID NO: 62, (j) The sgRNA targeting the Nanog promoter includes an RNA sequence corresponding to SEQ ID NO: 63, and / or (k) The composition according to claim 4, wherein the sgRNA targeting the EEA motif comprises an RNA sequence corresponding to Sequence ID No.

64.

6. The CRISPR activation system is, (a) an inactivated CRISPR-related nuclease fused with at least one transcription activator, (b) The composition according to any one of claims 1 to 5, comprising a tandem array of peptides linking an inactivated CRISPR-related nuclease to at least one transcription activator.

7. (a) The CRISPR activating system includes inactivated Cas9 (dCas9), (b) The tandem array of peptides is a SunTag array, (c) The composition according to claim 6, wherein the at least one transcription activator comprises herpes simplex VP16, a tetramer of VP16 (VP64), one or more acetyltransferase active domains of p300 (p300 core), or p65.

8. The aforementioned non-iPSC is (a) fibroblasts, skin cells, umbilical cord blood cells, peripheral blood cells, or renal epithelial cells, (b) Mammalian cells, and / or (c) The composition according to any one of claims 1 to 7, wherein the composition is human cells.

9. The composition according to any one of claims 1 to 8, comprising one or more small molecules including a TGFβR inhibitor, a GSK3 inhibitor, a MEK inhibitor, and a ROCK inhibitor.

10. A composition according to any one of claims 1 to 9, further comprising doxycycline.

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