CRISPR activation enables high fidelity reprogramming into human pluripotent stem cells (bulk cells)
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA682166 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR13182627_1.fastq.gz | Fastqsanger.gz | |||
| SRR13182627_2.fastq.gz | Fastqsanger.gz | |||
| SRR13182628_1.fastq.gz | Fastqsanger.gz | |||
| SRR13182628_2.fastq.gz | Fastqsanger.gz | |||
| SRR13182629_1.fastq.gz | Fastqsanger.gz |
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Project description:CRISPR activation enables high fidelity reprogramming into human pluripotent stem cells
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Project description:Conventional reprogramming methods rely on the ectopic expression of transcription factors to reprogram somatic cells into induced pluripotent stem cells (iPSC). Forced expression of transcription factors might lead to off-target gene activation and heterogeneous reprogramming, resulting in the emergence of alternative cell types and aberrant iPSC. Activation of endogenous pluripotency factors by CRISPR activation (CRISPRa) can potentially reduce this heterogeneity. Here we describe a high efficiency reprogramming of human somatic cells into iPSC using optimized CRISPRa. Efficient reprogramming was dependent on the additional targeting of the embryo genome activation-enriched Alu-motif and the miR-302/367 locus. Single-cell transcriptome analysis revealed that the optimized CRISPRa reprogrammed cells with more directly and specifically into the pluripotent state when compared to the conventional reprogramming method. These findings support the use of CRISPRa for high quality reprogramming of human iPSC.
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Project description:Conventional reprogramming methods rely on the ectopic expression of transcription factors to reprogram somatic cells into induced pluripotent stem cells (iPSC). Forced expression of transcription factors might lead to off-target gene activation and heterogeneous reprogramming, resulting in the emergence of alternative cell types and aberrant iPSC. Activation of endogenous pluripotency factors by CRISPR activation (CRISPRa) can potentially reduce this heterogeneity. Here we describe a high efficiency reprogramming of human somatic cells into iPSC using optimized CRISPRa. Efficient reprogramming was dependent on the additional targeting of the embryo genome activation-enriched Alu-motif and the miR-302/367 locus. Single-cell transcriptome analysis revealed that the optimized CRISPRa reprogrammed cells with more directly and specifically into the pluripotent state when compared to the conventional reprogramming method. These findings support the use of CRISPRa for high quality reprogramming of human iPSC. This SuperSeries is composed of the SubSeries listed below.
2021-12-23 | GSE162530 | GEO
Project description:Conventional reprogramming methods rely on the ectopic expression of transcription factors to reprogram somatic cells into induced pluripotent stem cells (iPSC). Forced expression of transcription factors might lead to off-target gene activation and heterogeneous reprogramming, resulting in the emergence of alternative cell types and aberrant iPSC. Activation of endogenous pluripotency factors by CRISPR activation (CRISPRa) can potentially reduce this heterogeneity. Here we describe a high efficiency reprogramming of human somatic cells into iPSC using optimized CRISPRa. Efficient reprogramming was dependent on the additional targeting of the embryo genome activation-enriched Alu-motif and the miR-302/367 locus. Single-cell transcriptome analysis revealed that the optimized CRISPRa reprogrammed cells with more directly and specifically into the pluripotent state when compared to the conventional reprogramming method. These findings support the use of CRISPRa for high quality reprogramming of human iPSC.
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Project description:CRISPR-Cas9-based gene activation (CRISPRa) is an attractive tool for cellular reprogramming applications due to its high multiplexing capacity and direct targeting of endogenous loci. Here we present the reprogramming of primary human skin fibroblasts into induced pluripotent stem cells (iPSCs) using CRISPRa, targeting endogenous OCT4, SOX2, KLF4, MYC, and LIN28A promoters. The low basal reprogramming efficiency can be improved by an order of magnitude by additionally targeting a conserved Alu-motif enriched near genes involved in embryo genome activation (EEA-motif). This effect is mediated in part by more efficient activation of NANOG and REX1. These data demonstrate that human somatic cells can be reprogrammed into iPSCs using only CRISPRa. Furthermore, the results unravel the involvement of EEA-motif-associated mechanisms in cellular reprogramming.
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Project description:Androgen deficiency is a common medical conditions that affects males of all ages. Transplantation of testosterone-producing cells is a promising treatment for male hypogonadism. However, getting a cell source with the characteristics of Leydig cells (LCs) is still a challenge. Here, a high-efficiency reprogramming of skin-derived fibroblasts into functional Leydig-like cells (LLCs) based on epigenetic mechanism was described. By performing an integrated analysis of genome-wide DNA methylation and transcriptome profiling in LCs and fibroblasts, the potentially epigenetic-regulating steroidogenic genes and signaling pathways were identified. Then by using CRISPR/dCas9 activation system and signaling pathway regulators, the male- or female-derived fibroblasts were reprogrammed into LLCs with main LC-specific traits. Transcriptomic analysis further indicated that the correlation coefficients of global genes and transcription factors between LLCs and LCs were higher than 0.81 and 0.96, respectively. After transplantation in the testes of hypogonadal rodent models, LLCs increased serum testosterone concentration significantly. In type 2 diabetic rats model, LLCs which were transplanted in armpit, have the capability to restore the serum testosterone level and improve the hyperglycemia status. In conclusion, our approach enables skin-derived fibroblasts reprogramming into LLCs with high fidelity, providing a potential cell source for the therapeutics of male hypogonadism and metabolic-related comorbidities.
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Project description:Pluripotent stem cells (PSCs) offer an exciting resource for probing human biology; however, gene-editing efficiency remains relatively low in many cell types, including stem cells. Gene-editing using the CRISPR-Cas9 system offers an attractive solution that improves upon previous gene-editing approaches; however, like other technologies, off-target mutagenesis remains a concern. High-fidelity Cas9 variants greatly reduce off-target mutagenesis and offer a solution to this problem. To evaluate their utility as part of a cell-based gene-editing platform, human PSC lines were generated with a high-fidelity (HF) tetracycline-inducible engineered Streptococcus pyogenes SpCas9 (HF-iCas9) integrated into the AAVS1 safe harbor locus. By engineering cells with controllable expression of Cas9, we eliminated the need to include a large Cas9-expressing plasmid during cell transfection. Delivery of genetic cargo was further optimized by packaging DNA targeting guide RNAs (gRNAs) and donor fragments into a single plasmid backbone. The potential of homology-directed repair (HDR) based gene knock-in at the CLYBL safe harbor site and endogenous SOX2 and SIX6 genes were demonstrated. Moreover, we used non-homologous end-joining (NHEJ) for gene knockout of disease-relevant alleles. These high-fidelity CRISPR tools and the resulting HF-iCas9 cell lines will facilitate the production of cell-type reporters and mutants across different genetic backgrounds.
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