Applying genome-wide CRISPR screens for therapeutic discovery in FSHD
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ABSTRACT:
PROVIDER: PRJNA551137 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR9602292.fastq.gz | Fastqsanger.gz | |||
| SRR9602293.fastq.gz | Fastqsanger.gz | |||
| SRR9602294.fastq.gz | Fastqsanger.gz | |||
| SRR9602295.fastq.gz | Fastqsanger.gz |
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Project description:The emergence of CRISPR gene-editing technologies and genome-wide CRISPR libraries enables a new mechanism to efficiently perform unbiased genetic screens that can accelerate the process of therapeutic discovery for genetic disorders. This represents a significant leap forward from the extensive studies of genetic and biochemical interactions typically required to understand disease pathogenesis and development of targeted therapy. In this study, we demonstrate the utility of a genome-wide CRISPR loss-of-function library to identify therapeutic targets for facioscapulohumeral muscular dystrophy (FSHD), a genetically complex subtype of muscular dystrophy for which there is currently no treatment available. In FSHD, both genetic and epigenetic changes can lead to misexpression of DUX4, the FSHD causal gene that encodes the highly cytotoxic DUX4 protein. We performed a genome-wide CRISPR screen to identify genes whose loss-of-function conferred survival when DUX4 was expressed, thus reducing the pathogenic impact of the genetic defect. Emerging genes from our screen illuminated ‘druggable’ pathways that can be targeted with small molecule inhibitors. Treatment with PI3K/Akt/mTOR signaling inhibitors resulted in reduced DUX4 protein expression and a reduction of FSHD disease biomarkers in patient myogenic lines, as well as improved structural and functional properties of a zebrafish model of FSHD. Our genome-wide perturbation of pathways affecting DUX4 expression has empowered us to rapidly identify existing compounds with potential therapeutic benefit for FSHD. Thus, the experimental pipeline described in this paper presents an accelerated paradigm towards therapeutic discovery and is translatable to other genetic diseases with well-established phenotypic selection assays.
2019-06-27 | GSE133332 | GEO
Project description:The emergence of CRISPR-Cas9 gene-editing technologies and genome-wide CRISPR-Cas9 libraries enables efficient unbiased genetic screening that can accelerate the process of therapeutic discovery for genetic disorders. Here, we demonstrate the utility of a genome-wide CRISPR-Cas9 loss-of-function library to identify therapeutic targets for facioscapulohumeral muscular dystrophy (FSHD), a genetically complex type of muscular dystrophy for which there is currently no treatment. In FSHD, both genetic and epigenetic changes lead to misexpression of DUX4, the FSHD causal gene that encodes the highly cytotoxic DUX4 protein. We performed a genome-wide CRISPR-Cas9 screen to identify genes whose loss-of-function conferred survival when DUX4 was expressed in muscle cells. Genes emerging from our screen illuminated a pathogenic link to the cellular hypoxia response, which was revealed to be the main driver of DUX4-induced cell death. Application of hypoxia signaling inhibitors resulted in increased DUX4 protein turnover and subsequent reduction of the cellular hypoxia response and cell death. In addition, these compounds proved successful in reducing FSHD disease biomarkers in patient myogenic lines, as well as improving structural and functional properties in two zebrafish models of FSHD. Our genome-wide perturbation of pathways affecting DUX4 expression has provided insight into key drivers of DUX4-induced pathogenesis and has identified existing compounds with potential therapeutic benefit for FSHD. Our experimental approach presents an accelerated paradigm toward mechanistic understanding and therapeutic discovery of a complex genetic disease, which may be translatable to other diseases with well-established phenotypic selection assays.
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Project description:Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disorder linked to contractions of the D4Z4 repeat array in the subtelomeric region of chromosome 4q. By comparing genome-wide gene expression data from muscle biopsies of patients with FSHD to those of 11 other neuromuscular disorders, we intend to identify disease-specific changes which are more likely to be involved in the early stages of the disease progression. The data will help to identify pathological mechanisms involved in FSHD. Keywords: Differentiation design
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Project description:Proteomic studies in facioscapulohumeral muscular dystrophy (FSHD) could offer new insight to disease mechanisms underpinned by post-transcriptional processes. We used stable isotope (deuterium oxide; D2O) labelling and peptide mass spectrometry to investigate the abundance and turnover rates of proteins in cultured muscle cells from 2 individuals affected by FSHD and their unaffected siblings (UASb). We measured the abundance of 4483 proteins and the turnover rate of 2324 proteins in each (n = 4) myoblast sample. FSHD myoblasts exhibited a greater abundance but slower turnover rate of subunits of mitochondrial respiratory complexes and mitochondrial ribosomal proteins, which may indicate an accumulation of ‘older’ less viable mitochondrial proteins in myoblasts from individuals affected by FSHD. Our results highlight the importance of post-transcriptional processes and protein turnover in FSHD pathology and provide a resource for the FSHD research community to explore this burgeoning aspect of FSHD.
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Project description:We performed CRISPR screens on both a sub-library and a genome-wide scale in human intestinal organoids to discover cancer driver genes. We investigated the Wnt and the TGFB pathway and used both WT, APC-mutant and APC-TP53-mutant organoids.
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Project description:The gene expression pathways leading to muscle pathology in facioscapulohumeral dystrophy (FSHD) remain to be elucidated. This muscular dystrophy is caused by a contraction of an array of tandem 3.3-kb repeats (D4Z4) at 4q35.2. We compared expression of control and FSHD myoblasts and myotubes (three preparations each) on exon microarrays (Affymetrix Human Exon 1.0 ST) and validated FSHD-specific differences for representative genes by qRT-PCR on additional myoblast cell strains. The FSHD and control myoblasts used for these experiments were shown to grow and differentiate into myotubes equally efficiently as control myoblasts. There were no significant FSHD-control differences in RNA levels for MYOD1 and MYOG at the myoblast and myotube stages and for MYF5 and MYF6 at the myoblast stage. In contrast, 295 other genes were dysregulated at least 2-fold in FSHD vs. control myoblasts (p <0.01, adjusted for multiple comparisons). Remarkably, only 10% of the FSHD-associated gene dysregulation at the myoblast stage was downregulation. At the myotube stage, about ten times as many genes exhibited FSHD-associated downregulated as at the myoblast stage and twice as many genes displayed FSHD-associated upregulation. The FSHD-related changes in RNA levels appear to be due to posttranscriptional as well as transcriptional alterations. Among the prominently dysregulated pathways were signaling and oxidative stress pathways. By comparing expression profiles of control myoblasts and myotubes to each other and to 19 non-muscle cell types profiled identically, our study also revealed many new myogenesis associations for genes not previously annotated as muscle-specific. Keywords: Disease state analysis and time course for differentiation
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