CRISPRa screen for drivers of COL7A1 expression in keratinocytes
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ABSTRACT:
PROVIDER: PRJNA684343 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR13239573.fastq.gz | Fastqsanger.gz | |||
| SRR13239574.fastq.gz | Fastqsanger.gz |
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Project description:Background: Dystrophic epidermolysis bullosa (DEB) is a skin blistering disease caused by mutations in COL7A1, which encodes type VII collagen (C7). There is no cure for DEB, but previous work has shown potential therapeutic benefit in increasing production of even partially functional C7. Genome-wide screens using CRISPR-Cas9 have enabled the identification of genes involved in cancer development, drug resistance, and other genetic diseases, suggesting that they could be used to identify novel drivers of C7 production. Methods: A keratinocyte C7 reporter cell line was created by integrating a tdTomato fluorescent marker into the last exon of the endogenous COL7A1 gene. A genome-wide CRISPR activation (CRISPRa) screen was performed with the C7_tdTomato reporter to identify genes and pathways that increase C7 expression. High tdTomato-expressing cells were sorted and sequenced to identify the single guide RNAs (sgRNAs) that became enriched relative to the starting material. Pathway analysis was performed on the corresponding genes to identify regulators and pathways that influence C7 expression. A targeted drug screen was performed in three different keratinocyte cell lines based on the CRISPRa screen results, and C7 upregulation was evaluated. Results: The C7_tdTomato cell line was validated as an effective reporter cell line for detection of C7 upregulation. The CRISPRa screen identified two genes, DENND4B and TYROBP as top hits based on enrichment in the high fluorescence population and representation with multiple sgRNAs. Pathway analysis of the CRISPRa screen showed enrichment of toll-like receptor and interferon-related upstream regulators and functions related to calcium uptake and immune signaling. Several compounds in the targeted drug screen increased C7 expression in at least one keratinocyte line, but only kaempferol, a plant flavonoid, significantly increased C7 mRNA and protein in a DEB patient line. Conclusions: The novel C7_tdTomato reporter cell line can be used to screen compounds for increased C7 expression. The CRISPRa screen combined with a fluorescent reporter cell line can be used to reveal mechanistic regulators of gene expression and therapeutic targets for rare genetic diseases. Kaempferol has shown promising results in increasing C7 production and should be further evaluated as a potential therapeutic for DEB patients.
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Project description:Patients with recessive dystrophic epidermolysis bullosa (RDEB) lack functional Type VII collagen and suffer severe blistering and chronic wounds that ultimately lead to infection and development of lethal squamous cell carcinoma (SCC). The discovery of induced pluripotent stem cells (iPSCs) and the ability to edit the genome bring the possibility to provide definitive genetic therapy through corrected autologous tissues. We have formed a multidisciplinary team with the ultimate goal to develop an iPSC-based therapy for RDEB. Here, we present a clinical protocol that generates autologous, corrected epithelial keratinocyte sheets with the COL7A1 gene mutation corrected for grafting on to patients. We demonstrate the utility of sequential reprogramming and novel adenovirus-associated viral genome editing to generate corrected iPSC banks. iPSC-derived keratinocytes were produced with minimal heterogeneity and secreted wild-type collagen VII, resulting in stratified epidermis in vitro and in vivo in mice. Sequencing of corrected cell lines prior to tissue formation revealed heterogeneity of SCC-predisposing mutations, allowing us to select COL7A1 corrected banks with minimal mutational burden for downstream epidermis production. Our results provide a first clinical platform to use iPSCs in the treatment of debilitating genodermatoses. Microarray analysis of iPS-derived keratinocytes from two RDEB patients (iPS-K1 and iPS-K3), corresponding patient keratinocytes (AHK1 and AHK2), normal human keratinocytes (NHK), as well as H9 human embryonic stem cells (hESC - H9). All samples were analyzed in duplicate and differential gene expression was measured relative to H9.
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Project description:We previously performed a tiling CRISPR activation (CRISPRa) screen in Jurkat T cells to discover enhancers controlling IL2RA expression. This screen identified six regions where recruitment of dCas9-VP64 was sufficient to drive increased expression of IL2RA. We named these putative enhancers CRISPRa Responsive Elements (CaREs). To examine transcriptome-wide consequences of dCas9-VP64 recruitment to IL2RA CaREs, we performed RNA-Seq on HuT78 cells stably expressing dCas9-VP64 and gRNAs targeting the IL2RA TSS, CaRE3, or CaRE4, or stimulated with anti-CD3/CD28 antibodies.
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Project description:Zygotic genome activation (ZGA) is a crucial developmental milestone that remains poorly understood. This first essential transcriptional event in embryonic development coincides with extensive epigenetic reprogramming and is orchestrated, in part, by the interplay of transcriptional and epigenetic regulators. Here, we developed a novel high-throughput screening method that combines pooled CRISPR-activation (CRISPRa) with single-cell transcriptomics and applied this method to systematically probe candidate regulators of ZGA-like transcription. We screened 230 epigenetic and transcriptional factors by upregulating their expression with CRISPRa in mouse embryonic stem cells (ESCs). Through single-cell RNA-sequencing (scRNA-seq), we generated approximately 200,000 single-cell transcriptomes of CRISPRa-perturbed cells, each transduced with a unique short-guide RNA (sgRNA) targeting a specific candidate gene promoter. Using multi-omics factor analysis (MOFA) of the perturbation scRNA-seq profiles, we characterized molecular signatures of ZGA and uncovered 24 factors that promote a ZGA-like response in ESCs, both in the coding and non-coding transcriptome. We further validated nine candidate genes by arrayed CRISPRa analysed by bulk transcriptomics, which demonstrates that the combination of CRISPRa with scRNA-seq is a powerful and valid approach to identify regulators of ZGA-like transcription. Additional cDNA overexpression assays for three top hits, the DNA binding protein Dppa2, the chromatin remodeller Smarca5 and the transcription factor Patz1, confirmed these factors as ZGA-like regulators by alternative methods. Supporting these findings, Dppa2 and Smarca5 knock-out ESCs lose expression of ZGA genes and functional experiments revealed that Smarca5’s regulation of ZGA-like transcription is dependent on Dppa2. Together, our single-cell transcriptomic profiling of CRISPRa-perturbed cells provides comprehensive system-level insights into the molecular mechanisms that orchestrate ZGA.
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Project description:Patients with recessive dystrophic epidermolysis bullosa (RDEB) lack functional Type VII collagen and suffer severe blistering and chronic wounds that ultimately lead to infection and development of lethal squamous cell carcinoma (SCC). The discovery of induced pluripotent stem cells (iPSCs) and the ability to edit the genome bring the possibility to provide definitive genetic therapy through corrected autologous tissues. We have formed a multidisciplinary team with the ultimate goal to develop an iPSC-based therapy for RDEB. Here, we present a clinical protocol that generates autologous, corrected epithelial keratinocyte sheets with the COL7A1 gene mutation corrected for grafting on to patients. We demonstrate the utility of sequential reprogramming and novel adenovirus-associated viral genome editing to generate corrected iPSC banks. iPSC-derived keratinocytes were produced with minimal heterogeneity and secreted wild-type collagen VII, resulting in stratified epidermis in vitro and in vivo in mice. Sequencing of corrected cell lines prior to tissue formation revealed heterogeneity of SCC-predisposing mutations, allowing us to select COL7A1 corrected banks with minimal mutational burden for downstream epidermis production. Our results provide a first clinical platform to use iPSCs in the treatment of debilitating genodermatoses.
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