Myogenic Progenitor Cell Lineage Specification by CRISPR/Cas9-based Transcriptional Activators
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE145575 | GEO | 2020/04/10
REPOSITORIES: GEO
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Project description:Technologies allowing for specific regulation of endogenous genes are valuable for the study of gene functions and have great potential in therapeutics. We created the CRISPR-on system, a two-component transcriptional activator consisting of a nuclease-dead Cas9 (dCas9) protein fused with a transcriptional activation domain and single guide RNAs (sgRNAs) with complementary sequence to gene promoters. We demonstrate that CRISPR-on can efficiently activate exogenous reporter genes in both human and mouse cells in a tunable manner. In addition, we show that robust reporter gene activation in vivo can be achieved by injecting the system components into mouse zygotes. Furthermore we show that CRISPR-on can activate the endogenous IL1RN, SOX2, and OCT4 genes. The most efficient gene activation was achieved by clusters of 3 to 4 sgRNAs binding to the proximal promoters suggesting their synergistic action in gene induction. Significantly, when sgRNAs targeting multiple genes were simultaneously introduced into cells, robust multiplexed endogenous gene activation was achieved. Genome-wide expression profiling demonstrated high specificity of the system. We used microarray to assay the gene expression changes after transfection of dCas9VP160 with the different sgRNAs
2013-08-10 | E-GEOD-49701 | biostudies-arrayexpress
Project description:Myogenic differentiation of iPSCs has been done by gene–overexpression or directed differentiation. However, viral integration, long-term culture and the presence of unwanted cells are the main obstacles. By using CRISPR/Cas9n, a novel double reporter hESC line was generated for PAX7/MYF5, allowing prospective readout. This strategy allowed pathway screen to define efficient myogenic induction in hESC/iPSCs. Next, surface marker screen allowed identification of CD10 and CD24 for purification of myogenic progenitors. CD10 expression was also confirmed on human satellite cells and muscle progenitors. In vivo studies using transgene/reporter-free hESC/iPSCs further validated myogenic potential of the cells by dystrophin restoration and satellite cell engraftment in NSG-mdx4cv mice. In addition, side-by-side in vitro and in vivo comparison proved superior specificity of CD10/CD24 compared to recently reported markers (ERBB3/NGFR). This study provides new biological insights for myogenic differentiation using a new cell resource and identifies CD10 as a potential myogenic marker.
2018-12-03 | GSE121154 | GEO
Project description:Myogenic Progenitor Cell Lineage Specification by CRISPR/Cas9-based Transcriptional Activators
| PRJNA607567 | ENA
Project description:Using dox-inducible mouse ES cells, we generated myogenic progenitors expressing the transcription factor Pax7. These cells were used for determining the genomic occupancy of the transcription factor Pax7.
2020-10-31 | GSE147057 | GEO
Project description:Skeletal myogenic commitment of human pluripotent cells can be achieved by doxycycline-inducible expression of the transcription factor PAX7. To gain further insights on PAX7 function during this process, we performed a time course whole transcriptome analysis of differentiating H9 human embryonic stem cells from doxycycline-treated and untreated cultures. In addition, we identified the genomic binding of PAX7 in one of the selected time point (referred as PAX7+ proliferating myogenic progenitors).
2017-06-30 | GSE98976 | GEO
Project description:Using Hi-C, promoter-capture Hi-C (pCHi-C), and other genome-wide approaches in skeletal muscle progenitors that inducibly express a master transcription factor, Pax7, we systematically characterized at high-resolution the spatio-temporal re-organization of compartments and promoter-anchored interactions as a consequence of myogenic commitment and differentiation. To further investigate transcriptional regulatory mechanisms governed by Pax7, we used immuno-affinity purification and mass spectrometric sequencing to identify the compendium of Pax7-associated proteins in muscle progenitors. Although Pax7-associated proteins have been identified in myoblasts, it is likely that such an approach would fail to uncover progenitor
specific interactions with this protein. We therefore isolated chromatin from iPax7 cells
engineered with a single, inducible copy of the Flag-tagged Pax7 transgene integrated next to the Hprt locus, which is expected to maintain expression levels comparable
to those of satellite cells in the presence of Dox. Through purification of Flag-Pax7, we
identified a cohort of factors and complexes involved in gene activation or repression and remodeling of chromatin and genome architecture that were substantially enriched compared to the uninduced control. We also identified a large cohort of sequence-specific TFs, including several that were shown to play an essential role in muscle stem cells (e.g., Foxk1, Six1, Runx1, Tead1, Nfix) and that are recruited to Pax7-bound enhancers in muscle cells. Importantly, we also confirmed multiple interactions from our proteomic screen through immunoprecipitation and western blotting.
2020-10-30 | MSV000086392 | MassIVE
Project description:Synthetic DNA-binding proteins have found broad application in gene therapies and as tools for interrogating biology. Engineered proteins based on the CRISPR/Cas9 and TALE systems have been used to alter genomic DNA sequences, control transcription of endogenous genes, and modify epigenetic states. Although the activity of these proteins at their intended genomic target sites have been assessed, the genome-wide effects of their action have not been extensively characterized. Additionally, the role of chromatin structure in determining the binding of CRISPR/Cas9 and TALE proteins to their target sites and the regulation of nearby genes is poorly understood. Characterization of the activity these proteins using modern high-throughput genomic methods would provide valuable insight into the specificity and off-target effects of CRISPR- and TALE-based genome engineering tools. We have analyzed the genome-wide effects of TALE- and CRISPR-based transcriptional activators targeted to the promoters of two different endogenous human genes in HEK293T cells using a variety of high-throughput DNA sequencing methods. In particular, we assayed the DNA-binding specificity of these proteins and their effects on the epigenome. DNA-binding specificity was evaluated by ChIP-seq and RNA-seq was used to measure the specificity of these activators in perturbing the transcriptome. Additionally, DNase-seq was used to identify the chromatin state at target sites of the synthetic transcriptional activators and the genome-wide chromatin remodeling that occurs as a result of their action. Our results show that these genome engineering technologies are highly specific in both binding to their promoter target sites and inducing expression of downstream genes when multiple activators bind to a single promoter. Moreover, we show that these synthetic activators are able to induce the expression of silent genes in heterochromatic regions of the genome by opening regions of closed chromatin and decreasing DNA methylation. Interestingly, the transcriptional activation domain was not necessary for DNA-binding or chromatin remodeling in these regions, but was critical to inducing gene expression. This study shows that these CRISPR- and TALE-based transcriptional activators are exceptionally specific. Although we detected limited binding of off-target sites in the genome and changes to genome structure, these off-target event did not lead to any detectable changes in gene regulation. Collectively, these results underscore the potential for these technologies to make precise changes to gene expression for gene and cell therapies or fundamental studies of gene function. HEK293T cells were transfected in triplicate with plasmids expressing synthetic transcription factors. The synthetic TFs were either (a) dCas9-VP64 fusion protein and a targeting guide RNA (gRNA), or (b) a TALE-VP64 fusion protein engineered to bind to a specific target site in the genome. As a control, cells were transfected with plasmids expressing GFP. After transfection, ChIP-seq was used to identify both on-target and off-target binding sites for the synthetic TFs.
2015-04-24 | E-GEOD-57085 | biostudies-arrayexpress
Project description:Synthetic DNA-binding proteins have found broad application in gene therapies and as tools for interrogating biology. Engineered proteins based on the CRISPR/Cas9 and TALE systems have been used to alter genomic DNA sequences, control transcription of endogenous genes, and modify epigenetic states. Although the activity of these proteins at their intended genomic target sites have been assessed, the genome-wide effects of their action have not been extensively characterized. Additionally, the role of chromatin structure in determining the binding of CRISPR/Cas9 and TALE proteins to their target sites and the regulation of nearby genes is poorly understood. Characterization of the activity these proteins using modern high-throughput genomic methods would provide valuable insight into the specificity and off-target effects of CRISPR- and TALE-based genome engineering tools. We have analyzed the genome-wide effects of TALE- and CRISPR-based transcriptional activators targeted to the promoters of two different endogenous human genes in HEK293T cells using a variety of high-throughput DNA sequencing methods. In particular, we assayed the DNA-binding specificity of these proteins and their effects on the epigenome. DNA-binding specificity was evaluated by ChIP-seq and RNA-seq was used to measure the specificity of these activators in perturbing the transcriptome. Additionally, DNase-seq was used to identify the chromatin state at target sites of the synthetic transcriptional activators and the genome-wide chromatin remodeling that occurs as a result of their action. Our results show that these genome engineering technologies are highly specific in both binding to their promoter target sites and inducing expression of downstream genes when multiple activators bind to a single promoter. Moreover, we show that these synthetic activators are able to induce the expression of silent genes in heterochromatic regions of the genome by opening regions of closed chromatin and decreasing DNA methylation. Interestingly, the transcriptional activation domain was not necessary for DNA-binding or chromatin remodeling in these regions, but was critical to inducing gene expression. This study shows that these CRISPR- and TALE-based transcriptional activators are exceptionally specific. Although we detected limited binding of off-target sites in the genome and changes to genome structure, these off-target event did not lead to any detectable changes in gene regulation. Collectively, these results underscore the potential for these technologies to make precise changes to gene expression for gene and cell therapies or fundamental studies of gene function. HEK293T cells were transfected in triplicate with plasmids expressing synthetic transcription factors. The synthetic TFs were either (a) dCas9-VP64 fusion protein and a targeting guide RNA (gRNA), or (b) a TALE-VP64 fusion protein engineered to bind to a specific target site in the genome. As a control, cells were transfected with plasmids expressing GFP. After transfection, RNA-seq was used to identify both on-target and off-target binding sites for the synthetic TFs. The data in this submission were generated using the TALE transfection experiments.
2015-05-27 | E-GEOD-68341 | biostudies-arrayexpress
Project description:Direct lineage reprogramming provides a unique system to study cell fate transitions and unearth molecular mechanisms that safeguard cellular identity. We previously reported on direct conversion of mouse fibroblasts into induced myogenic progenitor cells (iMPCs) by means of transient MyoD overexpression in concert with small molecules treatment. Here we employed integrative multi-omic assays to delineate the molecular landscape of fibroblast reprogramming into iMPCs in comparison to transdifferentiation into myogenic cells solely by MyoD overexpression. Utilizing bulk RNA-sequencing and mass spectrometry we characterized molecular regulators and pathways that endow a myogenic stem cell identity on fibroblasts only in the presence of small molecule treatment. We further demonstrate that iMPC reprogramming is a stepwise process, commencing with the appearance of myofibers and committed myogenic progenitors prior to the formation of satellite cell-like myogenic progenitors that express a suite of stem cell markers including Pax7, Dek, Myf5, Sox8, and Dmrt2. To directly compare iMPCs to satellite cell-derived primary myoblasts, we employed a fluorescent Pax7-GFP reporter to purify Pax7+ cells from the heterogenous iMPC cultures and assess their equivalency to FACS-purified Pax7-GFP+ myoblasts. We demonstrate that Pax7+ iMPCs share molecular attributes with myoblasts, however also express genes and signaling pathways that are indicative of activated satellite cells including Carm1, Dlk1, Lgr5, Fos, Dek, Calcr and Pitx3. We further establish that iMPC formation and maintenance is dependent on the Notch pathway, as small molecule inhibition of Notch abrogates iMPC formation and derails stable iMPC cultures via depletion of Pax7 expressing cells. Lastly, using single cell RNA-sequencing we determine the cell populations that comprise iMPCs in addition to reconstructing the differentiation trajectory present in these heterogenous cultures. We demonstrate that a highly proliferative activated satellite cell-like population differentiates into committed myoblasts and myocytes that further give rise to myofibers that express mature muscle markers, thus capturing a dynamic differentiation program in vitro. Collectively, this study charts a molecular blueprint for reprogramming fibroblasts into myogenic stem and progenitor cells and further establishes the fidelity of stable iMPC cultures in capturing skeletal muscle regeneration in vitro for disease modeling and basic research applications.
2022-01-06 | GSE186271 | GEO
Project description:Direct lineage reprogramming provides a unique system to study cell fate transitions and unearth molecular mechanisms that safeguard cellular identity. We previously reported on direct conversion of mouse fibroblasts into induced myogenic progenitor cells (iMPCs) by means of transient MyoD overexpression in concert with small molecules treatment. Here we employed integrative multi-omic assays to delineate the molecular landscape of fibroblast reprogramming into iMPCs in comparison to transdifferentiation into myogenic cells solely by MyoD overexpression. Utilizing bulk RNA-sequencing and mass spectrometry we characterized molecular regulators and pathways that endow a myogenic stem cell identity on fibroblasts only in the presence of small molecule treatment. We further demonstrate that iMPC reprogramming is a stepwise process, commencing with the appearance of myofibers and committed myogenic progenitors prior to the formation of satellite cell-like myogenic progenitors that express a suite of stem cell markers including Pax7, Dek, Myf5, Sox8, and Dmrt2. To directly compare iMPCs to satellite cell-derived primary myoblasts, we employed a fluorescent Pax7-GFP reporter to purify Pax7+ cells from the heterogenous iMPC cultures and assess their equivalency to FACS-purified Pax7-GFP+ myoblasts. We demonstrate that Pax7+ iMPCs share molecular attributes with myoblasts, however also express genes and signaling pathways that are indicative of activated satellite cells including Carm1, Dlk1, Lgr5, Fos, Dek, Calcr and Pitx3. We further establish that iMPC formation and maintenance is dependent on the Notch pathway, as small molecule inhibition of Notch abrogates iMPC formation and derails stable iMPC cultures via depletion of Pax7 expressing cells. Lastly, using single cell RNA-sequencing we determine the cell populations that comprise iMPCs in addition to reconstructing the differentiation trajectory present in these heterogenous cultures. We demonstrate that a highly proliferative activated satellite cell-like population differentiates into committed myoblasts and myocytes that further give rise to myofibers that express mature muscle markers, thus capturing a dynamic differentiation program in vitro. Collectively, this study charts a molecular blueprint for reprogramming fibroblasts into myogenic stem and progenitor cells and further establishes the fidelity of stable iMPC cultures in capturing skeletal muscle regeneration in vitro for disease modeling and basic research applications.
2022-01-06 | GSE169054 | GEO