scholarly journals A genome-wide CRISPR/Cas phenotypic screen for modulators of DUX4 cytotoxicity reveals screen complications

2020 ◽  
Author(s):  
Ator Ashoti ◽  
Francesco Limone ◽  
Melissa van Kranenburg ◽  
Anna Alemany ◽  
Mirna Baak ◽  
...  
Keyword(s):  
Treatment Options ◽  
Underlying Mechanism ◽  
Facioscapulohumeral Muscular Dystrophy ◽  
Knock Out ◽  
Genome Wide ◽  
Phenotypic Screen ◽  
A Genome ◽  
The Face ◽  
Muscle Disorder ◽  
Pioneer Transcription Factor

AbstractFacioscapulohumeral muscular dystrophy (FHSD), a fundamentally complex muscle disorder that thus far remains untreatable. As the name implies, FSHD starts in the muscles of the face and shoulder gridle. The main perturbator of the disease is the pioneer transcription factor DUX4, which is misexpressed in affected tissues due to a failure in epigenetic repressive mechanisms. In pursuit of unraveling the underlying mechanism of FSHD and finding potential therapeutic targets or treatment options, we performed an exhaustive genome-wide CRISPR/Cas9 phenotypic rescue screen to identify modulators of DUX4 cytotoxicity. We found no key effectors other than DUX4 itself, suggesting treatment efforts in FSHD should be directed towards its direct modulation.The screen did however reveal some rare and unexpected Cas9-induced genomic events, that may provide important considerations for planning future CRISPR/Cas9 knock-out screens.

BIOM-32. ENDOPLASMIC RETICULUM PROTEIN SSR3 DETERMINES AND PREDICTS RESPONSE TO PACLITAXEL IN BREAST CANCER AND GLIOBLASTOMA

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi17-vi18
Author(s):  
Crismita Dmello ◽  
Aarón Sonabend ◽  
Víctor Arrieta ◽  
Daniel Zhang ◽  
Deepak Kanojia ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Endoplasmic Reticulum ◽  
Glioma Cells ◽  
Predictive Biomarkers ◽  
Predictive Biomarker ◽  
Precision Oncology ◽  
Patient Response ◽  
Knock Out ◽  
Genome Wide ◽  
A Genome

Abstract Paclitaxel (PTX) is one the most potent and commonly used chemotherapies for breast and pancreatic cancer. Given the potency of this drug for glioblastomas (GBM) several ongoing clinical trials are investigating means of enhancing delivery of PTX across the blood-brain barrier for this disease. In spite of the efficacy of PTX, individual tumors exhibit variable susceptibility to this drug, with response rate in the range of 30%-60%. To identify predictive biomarkers for response to PTX, we performed a genome-wide CRISPR knock-out screen using human glioma cells. The most enriched genes in the CRISPR screen underwent further selection based on their correlation with survival in the breast cancer patient cohorts treated with PTX and not in patients treated with other chemotherapies, a finding that was validated on a second independent patient cohort. This led to the discovery of endoplasmic reticulum (ER) protein SSR3 as a putative predictive biomarker for PTX. SSR3 protein levels showed positive correlation with response to PTX in breast cancer cells, glioma cells, in multiple intracranial glioma xenografts and in GBM patient derived explant cultures. Knockout of SSR3 turned the cells resistant to PTX while its overexpression sensitized the cells to PTX. In gliomas, SSR3-mediated susceptibility to PTX relates to modulation of phosphorylation of ER stress sensor IRE1α. Thus, by using genome-wide screen combined with patient response data, we discovered a biomarker that demonstrates causal and correlative relationship with response to PTX in breast cancer and GBM. Prospective validation of this biomarker is warranted for its broad implementation for precision oncology.

Phenotypic Screen and Transcriptomics Approach Complement Each Other in Functional Genomics of Defensive Stink Gland Physiology

2021 ◽  
Author(s):  
Sabrina Lehmann ◽  
Bibi Atika ◽  
Daniela Grossmann ◽  
Christian Schmitt-Engel ◽  
Nadi Strohlein ◽  
...  
Keyword(s):  
Functional Genomics ◽  
Large Scale ◽  
Reverse Genetics ◽  
Expression Profiles ◽  
Forward Genetics ◽  
Large Set ◽  
Knock Down ◽  
Genome Wide ◽  
Phenotypic Screen ◽  
A Genome

Abstract Background Functional genomics uses unbiased systematic genome-wide gene disruption or analyzes natural variations such as gene expression profiles of different tissues from multicellular organisms to link gene functions to particular phenotypes. Functional genomics approaches are of particular importance to identify large sets of genes that are specifically important for a particular biological process beyond known candidate genes, or when the process has not been studied with genetic methods before. Results Here, we present a large set of genes whose disruption interferes with the function of the odoriferous defensive stink glands of the red flour beetle Tribolium castaneum. This gene set is the result of a large-scale systematic phenotypic screen using a reverse genetics strategy based on RNA interference applied in a genome-wide forward genetics manner. In this first-pass screen, 130 genes were identified, of which 69 genes could be confirmed to cause knock-down gland phenotypes, which vary from necrotic tissue and irregular reservoir size to irregular color or separation of the secreted gland compounds. The knock-down of 13 genes caused specifically a strong reduction of para-benzoquinones, suggesting a specific function in the synthesis of these toxic compounds. Only 14 of the 69 confirmed gland genes are differentially overexpressed in stink gland tissue and thus could have been detected in a transcriptome-based analysis. Moreover, of the 29 previously transcriptomics-identified genes causing a gland phenotype, only one gene was recognized by this phenotypic screen despite the fact that 13 of them were covered by the screen. Conclusion Our results indicate the importance of combining diverse and independent methodologies to identify genes necessary for the function of a certain biological tissue, as the different approaches do not deliver redundant results but rather complement each other. The presented phenotypic screen together with a transcriptomics approach are now providing a set of close to hundred genes important for odoriferous defensive stink gland physiology in beetles.

scholarly journals Identification of novel factors involved in or regulating initiation of DNA replication by a genome-wide phenotypic screen inSaccharomyces cerevisiae

Cell Cycle ◽  
2010 ◽  
Vol 9 (21) ◽  
pp. 4399-4410 ◽  
Author(s):  
Lijuan Ma ◽  
Yuanliang Zhai ◽  
Daorong Feng ◽  
Tsz-choi Chan ◽  
Yongjun Lu ◽  
...  
Keyword(s):  
Dna Replication ◽  
Genome Wide ◽  
Phenotypic Screen ◽  
A Genome ◽  
Initiation Of Dna Replication

scholarly journals Predicting the Landscape of Recombination Using Deep Learning

2020 ◽  
Vol 37 (6) ◽  
pp. 1790-1808 ◽  
Author(s):  
Jeffrey R Adrion ◽  
Jared G Galloway ◽  
Andrew D Kern
Keyword(s):  
Deep Learning ◽  
Evolutionary History ◽  
Model Misspecification ◽  
Natural Populations ◽  
High Accuracy ◽  
Demographic Model ◽  
Recombination Rates ◽  
Genome Wide ◽  
A Genome ◽  
The Face

Abstract Accurately inferring the genome-wide landscape of recombination rates in natural populations is a central aim in genomics, as patterns of linkage influence everything from genetic mapping to understanding evolutionary history. Here, we describe recombination landscape estimation using recurrent neural networks (ReLERNN), a deep learning method for estimating a genome-wide recombination map that is accurate even with small numbers of pooled or individually sequenced genomes. Rather than use summaries of linkage disequilibrium as its input, ReLERNN takes columns from a genotype alignment, which are then modeled as a sequence across the genome using a recurrent neural network. We demonstrate that ReLERNN improves accuracy and reduces bias relative to existing methods and maintains high accuracy in the face of demographic model misspecification, missing genotype calls, and genome inaccessibility. We apply ReLERNN to natural populations of African Drosophila melanogaster and show that genome-wide recombination landscapes, although largely correlated among populations, exhibit important population-specific differences. Lastly, we connect the inferred patterns of recombination with the frequencies of major inversions segregating in natural Drosophila populations.

scholarly journals Population Genomic Evidence Reveals Subtle Patterns of Differentiation in the Trophically Polymorphic Cuatro Ciénegas Cichlid, Herichthys minckleyi

2019 ◽  
Vol 110 (3) ◽  
pp. 361-369 ◽  
Author(s):  
Katherine L Bell ◽  
Chris C Nice ◽  
Darrin Hulsey
Keyword(s):  
Gene Flow ◽  
Biological Diversity ◽  
Molecular Genetic ◽  
Molecular Ecology ◽  
Cichlid Fish ◽  
Genome Wide ◽  
A Genome ◽  
The Face ◽  
Or Gene ◽  
Herichthys Minckleyi

Abstract In recent decades, an increased understanding of molecular ecology has led to a reinterpretation of the role of gene flow during the evolution of reproductive isolation and biological novelty. For example, even in the face of ongoing gene flow strong selection may maintain divergent polymorphisms, or gene flow may introduce novel biological diversity via hybridization and introgression from a divergent species. Herein, we elucidate the evolutionary history and genomic basis of a trophically polymorphic trait in a species of cichlid fish, Herichthys minckleyi. We explored genetic variation at 3 hierarchical levels; between H. minckleyi (n = 69) and a closely related species Herichthys cyanoguttatus (n = 10), between H. minckleyi individuals from 2 geographic locations, and finally between individuals with alternate morphotypes at both a genome-wide and locus-specific scale. We found limited support for the hypothesis that the H. minckleyi polymorphism is the result of ongoing hybridization between the 2 species. Within H. minckleyi we found evidence of geographic genetic structure, and using traditional population genetic analyses found that individuals of alternate morphotypes within a pool appear to be panmictic. However, when we used a locus-specific approach to examine the relationship between multi-locus genotype, tooth size, and geographic sampling, we found the first evidence for molecular genetic differences between the H. minckleyi morphotypes.

scholarly journals Applying genome-wide CRISPR-Cas9 screens for therapeutic discovery in facioscapulohumeral muscular dystrophy

2020 ◽  
Vol 12 (536) ◽  
pp. eaay0271 ◽  
Author(s):  
Angela Lek ◽  
Yuanfan Zhang ◽  
Keryn G. Woodman ◽  
Shushu Huang ◽  
Alec M. DeSimone ◽  
...  
Keyword(s):  
Cell Death ◽  
Muscular Dystrophy ◽  
Phenotypic Selection ◽  
Facioscapulohumeral Muscular Dystrophy ◽  
Loss Of Function ◽  
Hypoxia Response ◽  
Genome Wide ◽  
A Genome ◽  
Structural And Functional Properties ◽  
Cellular Hypoxia

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.

scholarly journals Japan COVID-19 Task Force: a nation-wide consortium to elucidate host genetics of COVID-19 pandemic in Japan

2021 ◽  
Author(s):  
Ho Namkoong ◽  
Ryuya Edahiro ◽  
Koichi Fukunaga ◽  
Yuya Shirai ◽  
Kyuto Sonehara ◽  
...  
Keyword(s):  
Genome Wide Association Study ◽  
Risk Allele ◽  
Task Force ◽  
European Population ◽  
P Value ◽  
Host Genetics ◽  
Risk Variants ◽  
Genome Wide ◽  
A Genome ◽  
The Face

To elucidate the host genetic loci affecting severity of SARS-CoV-2 infection, or Coronavirus disease 2019 (COVID-19), is an emerging issue in the face of the current devastating pandemic. Here, we report a genome-wide association study (GWAS) of COVID-19 in a Japanese population led by the Japan COVID-19 Task Force, as one of the initial discovery GWAS studies performed on a non-European population. Enrolling a total of 2,393 cases and 3,289 controls, we not only replicated previously reported COVID-19 risk variants (e.g., LZTFL1, FOXP4, ABO, and IFNAR2), but also found a variant on 5p35 (rs60200309-A at DOCK2) that was significantly associated with severe COVID-19 in younger (<65 years of age) patients with a genome-wide significant p-value of 1.2 × 10-8 (odds ratio = 2.01, 95% confidence interval = 1.58-2.55). This risk allele was prevalent in East Asians, including Japanese (minor allele frequency [MAF] = 0.097), but rarely found in Europeans. Cross-population Mendelian randomization analysis made a causal inference of a number of complex human traits on COVID-19. In particular, obesity had a significant impact on severe COVID-19. The presence of the population-specific risk allele underscores the need of non-European studies of COVID-19 host genetics.

scholarly journals The E3 ligase UBR2 regulates cell death under caspase deficiency via Erk/MAPK pathway

2020 ◽  
Vol 11 (12) ◽  
Author(s):  
Elodie Villa ◽  
Rachel Paul ◽  
Ophélie Meynet ◽  
Sophie Volturo ◽  
Guillaume Pinna ◽  
...  
Keyword(s):  
Cell Death ◽  
Mapk Pathway ◽  
Underlying Mechanism ◽  
Erk Pathway ◽  
Breast Cancers ◽  
Cell Death Pathway ◽  
Main Cell ◽  
Caspase Inhibition ◽  
Genome Wide ◽  
A Genome

AbstractEscape from cell death is a key event in cancer establishment/progression. While apoptosis is often considered as the main cell death pathway, upon caspase inhibition, cell death is rather delayed than blocked leading to caspase-independent cell death (CICD). Although described for years, CICD’s underlying mechanism remains to be identified. Here, we performed a genome-wide siRNA lethality screening and identified the RING-Type E3 Ubiquitin Transferase (UBR2) as a specific regulator of CICD. Strikingly, UBR2 downregulation sensitized cells towards CICD while its overexpression was protective. We established that UBR2-dependent protection from CICD was mediated by the MAPK/Erk pathway. We then observed that UBR2 is overexpressed in several cancers, especially in breast cancers and contributes to CICD resistance. Therefore, our work defines UBR2 as a novel regulator of CICD, found overexpressed in cancer cells, suggesting that its targeting may represent an innovative way to kill tumor cells.

scholarly journals Genome-wide CRISPR-based gene knockout screens reveal cellular factors and pathways essential for nasopharyngeal carcinoma

2019 ◽  
Vol 294 (25) ◽  
pp. 9734-9745 ◽  
Author(s):  
Chong Wang ◽  
Sizun Jiang ◽  
Liangru Ke ◽  
Luyao Zhang ◽  
Difei Li ◽  
...  
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Advanced Disease ◽  
Gene Knockout ◽  
Histone Acetyl Transferase ◽  
Knock Out ◽  
Genome Wide ◽  
A Genome ◽  
Cellular Factors ◽  
New Treatments ◽  
Specific Symptoms

Early diagnosis of nasopharyngeal carcinoma (NPC) is difficult because of a lack of specific symptoms. Many patients have advanced disease at diagnosis, and these patients respond poorly to treatment. New treatments are therefore needed to improve the outcome of NPC. To better understand the molecular pathogenesis of NPC, here we used an NPC cell line in a genome-wide CRISPR-based knockout screen to identify the cellular factors and pathways essential for NPC (i.e. dependence factors). This screen identified the Moz, Ybf2/Sas3, Sas2, Tip60 histone acetyl transferase complex, NF-κB signaling, purine synthesis, and linear ubiquitination pathways; and MDM2 proto-oncogene as NPC dependence factors/pathways. Using gene knock out, complementary DNA rescue, and inhibitor assays, we found that perturbation of these pathways greatly reduces the growth of NPC cell lines but does not affect growth of SV40-immortalized normal nasopharyngeal epithelial cells. These results suggest that targeting these pathways/proteins may hold promise for achieving better treatment of patients with NPC.

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