Homo sapiens
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA203652 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR863226.fastq.gz | Fastqsanger.gz | |||
| SRR863227.fastq.gz | Fastqsanger.gz | |||
| SRR863228.fastq.gz | Fastqsanger.gz | |||
| SRR863229.fastq.gz | Fastqsanger.gz | |||
| SRR863230.fastq.gz | Fastqsanger.gz |
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Project description:Identifying putative transcription factor target genes by combining CRISPR/Cas9-based transcriptional activation with RNAseq in Drosophila S2R+ cells. This study focuses on the transcription factors Twist and Snail, singly and together. RNA from Drosophila cells following CRISPR/Cas9-based activation of Twist, Snail, or Twist and Snail together, compared with non-targeting sgRNA. Two biological replicates for each experiment
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Project description:Engineered CRISPR/Cas9-based transcriptional activators can potently and specifically activate endogenous fate-determining genes to direct differentiation of pluripotent stem cells. Here, we demonstrate that endogenous activation of the PAX7 transcription factor results in stable epigenetic remodeling and directly reprograms human pluripotent stem cells into skeletal myoblast progenitor cells. Compared to the exogenous overexpression of PAX7 cDNA, we find that endogenous activation results in the generation of more proliferative myogenic progenitors that can maintain PAX7 expression over multiple passages in serum-free conditions while preserving the capacity for terminal myogenic differentiation. Transplantation of human myogenic progenitors derived from endogenous activation of PAX7 into immunodeficient mice resulted in a greater number of human dystrophin+ myofibers compared to exogenous PAX7 overexpression. RNA-seq analysis also revealed transcriptome-wide differences between myogenic progenitors generated via CRISPR-based endogenous activation of PAX7 and exogenous PAX7 cDNA overexpression. These studies demonstrate the utility of CRISPR/Cas9-based transcriptional activators for progenitor cell specification and their potential for regenerative medicine.
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Project description:Integration of the HIV-1 provirus in the host genome ensures a persistent supply of latently infected cells. This latent reservoir is recalcitrant to antiretroviral therapy (ART) making lifelong treatment the only option for patients. The â??shock and killâ?? strategy aims to eradicate latent HIV by reactivating proviral gene expression followed by ART treatment. Gene specific transcriptional activation can be achieved using the RNA-guided CRISPR-Cas9 system comprising small guide RNAs (sgRNAs) with a nuclease deficient Cas9 mutant (dCas9) fused to the VP64 transactivation domain (dCas9-VP64).  We engineered this system to target 23 sites within the LTR promoter of HIV-1 and identified a â??hotspotâ?? for activation. We studied the functionality of activating sgRNAs to transcriptionally modulate the latent proviral genome across multiple different in vitro latency cell models including several J-Lat, ACH2 J1.1 and the CEM T cell model comprising a single clonal population of integrated mCherry-IRES-Tat from a full-length HIV LTR (LChIT). While we observed variable responses of latent cell models to well-characterized chemical stimuli, we detected consistent efficient activation of latent virus mediated by activator sgRNAs. In addition, transcriptome analysis of chemically treated cells revealed massive non-specific gene dysregulation whereas by comparison, dCas9-VP64/sgRNAs induced specific activation of the integrated provirus. In conclusion, we show the potential for CRISPR-mediated gene activation systems to provide enhanced efficiency and specificity in a targeted latency reactivation strategy. This represents a promising approach to a â??functional cureâ?? of HIV/AIDS. Three experimental conditions (sgRNA control, TNF treated and sgRNA against the LTR of HIV-1) were analyzed in triplicate using two sequencing lanes
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Project description:We established human colorectal tumor organoids from benign adenoma, primary colorectal cancer or metastasized colorectal cancer. The gene signature of tumor organoids associated with their tumor progression status. We also generated genome-edited organoids from human intestinal organoids recapitulating adenoma-carcinoma sequence. Gene expression signature of the genome engineered organoids were similar to that of adenoma organoids. This result indicated multiple (up to five) genetic mutations were insufficient for gene expression reprogramming of colorectal cancer. We used microarrays to detail the global program of gene expression in human colorectal tumor organoids and artificially mutation introduced organoids. To assess the expression profiling of genome-engineered organoids, we prepared total-RNA from cultured adenoma, carcinoma and genome-engineered organoids. We produced two types of genome-engineered organoids using the CRISPR/Cas9 or lentivirus vector system. Each engineered gene and engineered methods are described as a single alphabet and method name, respectively, in the sample characteristics field. The abbreviations for the engineered genes are as follows. 1) Genome-engineered organoids with CRISPR/Cas9 A = APC deletion; K = KRAS G12V knock in; S = Smad4 deletion; T = TP53 deletion; P = PIK3CA E545K knock in. 2) Genome-engineered organoids with Lent virus vector B = CTNNB1 S33Y overexpression; K = KRAS G12V overexpression; S = Smad4 shRNA overexpression; T = TP53 shRNA overexpression; P = PIK3CA E545K overexpression.
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Project description:Identifying putative transcription factor target genes by combining CRISPR/Cas9-based transcriptional activation with RNAseq in Drosophila S2R+ cells. This study focuses on the transcription factors Twist and Snail, singly and together.
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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.
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Project description:New, orthogonal transcription factors in eukaryotic cells have been realized by engineering nuclease-deficient CRISPR-associated proteins and/or their guide RNAs. In this work, we present a new kind of orthogonal activators, in Saccharomyces cerevisiae, made by turning type V CRISPR RNA into a scaffold RNA (ScRNA) able to recruit a variable number of VP64 activation domains. The activator arises from the complex between the synthetic ScRNA and DNase-deficient type V Cas proteins: dCas12e and denAsCas12a. The transcription activation achieved via the newly engineered dCas:ScRNA system is up to 4.7-fold higher than that obtained with the direct fusion of VP64 to Cas proteins. The new transcription factors have been proven to be functional in circuits such as Boolean gates, converters, multiplex-gene and metabolic-pathway activation. Our results extend the CRISPR-Cas-based technology with a new effective tool that only demands RNA engineering and improves the current design of transcription factors based on type V Cas proteins.
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