Genome-wide CRISPR screen identifies protein pathways modulating tau protein levels in neurons

AbstractAggregates of hyperphosphorylated tau protein are a pathological hallmark of more than 20 distinct neurodegenerative diseases, including Alzheimer’s disease, progressive supranuclear palsy, and frontotemporal dementia. While the exact mechanism of tau aggregation is unknown, the accumulation of aggregates correlates with disease progression. Here we report a genome-wide CRISPR screen to identify modulators of endogenous tau protein for the first time. Primary screens performed in SH-SY5Y cells, identified positive and negative regulators of tau protein levels. Hit validation of the top 43 candidate genes was performed using Ngn2-induced human cortical excitatory neurons. Using this approach, genes and pathways involved in modulation of endogenous tau levels were identified, including chromatin modifying enzymes, neddylation and ubiquitin pathway members, and components of the mTOR pathway. TSC1, a critical component of the mTOR pathway, was further validated in vivo, demonstrating the relevance of this screening strategy. These findings may have implications for treating neurodegenerative diseases in the future.

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In vivo CRISPR/Cas9 knockout screen: TCEAL1 silencing enhances docetaxel efficacy in prostate cancer

Life Science Alliance ◽

10.26508/lsa.202000770 ◽

2020 ◽

Vol 3 (12) ◽

pp. e202000770 ◽

Cited By ~ 1

Author(s):

Linda K Rushworth ◽

Victoria Harle ◽

Peter Repiscak ◽

William Clark ◽

Robin Shaw ◽

...

Keyword(s):

Prostate Cancer ◽

Elongation Factor ◽

Enrichment Analysis ◽

Gene Set Enrichment Analysis ◽

Genome Wide ◽

A Genome ◽

Docetaxel Treatment ◽

Crispr Screen ◽

Altered Cell

Docetaxel chemotherapy in metastatic prostate cancer offers only a modest survival benefit because of emerging resistance. To identify candidate therapeutic gene targets, we applied a murine prostate cancer orthograft model that recapitulates clinical invasive prostate cancer in a genome-wide CRISPR/Cas9 screen under docetaxel treatment pressure. We identified 17 candidate genes whose suppression may enhance the efficacy of docetaxel, with transcription elongation factor A–like 1 (Tceal1) as the top candidate. TCEAL1 function is not fully characterised; it may modulate transcription in a promoter dependent fashion. Suppressed TCEAL1 expression in multiple human prostate cancer cell lines enhanced therapeutic response to docetaxel. Based on gene set enrichment analysis from transcriptomic data and flow cytometry, we confirmed that loss of TCEAL1 in combination with docetaxel leads to an altered cell cycle profile compared with docetaxel alone, with increased subG1 cell death and increased polyploidy. Here, we report the first in vivo genome-wide treatment sensitisation CRISPR screen in prostate cancer, and present proof of concept data on TCEAL1 as a candidate for a combinational strategy with the use of docetaxel.

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Small molecule v-ATPase inhibitor Etidronate lowers levels of ALS protein ataxin-2

10.1101/2021.12.20.473567 ◽

2021 ◽

Author(s):

Garam Kim ◽

Lisa Nakayama ◽

Jacob A Blum ◽

Tetsuya Akiyama ◽

Steven Boeynaems ◽

...

Keyword(s):

Spinocerebellar Ataxia Type ◽

Atpase Inhibitor ◽

Protein Levels ◽

Genome Wide ◽

A Genome ◽

Human Neurons ◽

Crispr Screen ◽

Ataxin 2 ◽

Antisense Oligonucleotide Therapy

Antisense oligonucleotide therapy targeting ATXN2, a gene in which mutations cause neurodegenerative diseases spinocerebellar ataxia type 2 and amyotrophic lateral sclerosis, has entered clinical trials in humans. Additional methods to lower ataxin 2 levels would be beneficial not only in uncovering potentially cheaper or less invasive therapies, but also in gaining greater mechanistic insight into how ataxin 2 is normally regulated. We performed a genome-wide fluorescence activated cell sorting (FACS)-based CRISPR screen in human cells and identified multiple subunits of the lysosomal vacuolar ATPase (v ATPase) as regulators of ataxin 2 levels. We demonstrate that Etidronate, a U.S. Food and Drug Administration (FDA)-approved drug that inhibits the v ATPase, lowers ataxin 2 protein levels in mouse and human neurons. Moreover, oral administration of the drug to mice in their water supply and food is sufficient to lower ataxin-2 levels in the brain. Thus, we uncover Etidronate as a safe and inexpensive compound for lowering ataxin-2 levels and demonstrate the utility of FACS-based screens for identifying targets to modulate levels of human disease proteins.

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A genome-wide CRISPR screen identifies the glycosylation enzyme DPM1 as a modifier of DPAGT1 deficiency and ER stress

10.1101/2021.12.03.471178 ◽

2021 ◽

Author(s):

Hans M. Dalton ◽

Raghuvir Viswanatha ◽

Ricky Brathwaite ◽

Jae Sophia Zuno ◽

Stephanie E. Mohr ◽

...

Keyword(s):

Er Stress ◽

Therapeutic Targets ◽

Therapeutic Benefit ◽

Loss Of Function ◽

Gene Encoding ◽

Fructose Metabolism ◽

Genome Wide ◽

A Genome ◽

Crispr Screen

AbstractPartial loss-of-function mutations in glycosylation pathways underlie a set of rare diseases called Congenital Disorders of Glycosylation (CDGs). In particular, DPAGT1-CDG is caused by mutations in the gene encoding the first step in N-glycosylation, DPAGT1, and this disorder currently lacks effective therapies. To identify potential therapeutic targets for DPAGT1-CDG, we performed CRISPR knockout screens in Drosophila cells for genes associated with better survival and glycoprotein levels under DPAGT1 inhibition. We identified hundreds of candidate genes that may be of therapeutic benefit. Intriguingly, inhibition of the mannosyltransferase Dpm1, or its downstream glycosylation pathways, could rescue two in vivo models of DPAGT1 inhibition and ER stress, even though impairment of these pathways alone usually cause CDGs. While both in vivo models ostensibly cause ER stress (through DPAGT1 inhibition or a misfolded protein), we found a novel difference in fructose metabolism that may indicate glycolysis as a modulator of DPAGT1-CDG. Our results provide new therapeutic targets for DPAGT1-CDG, include the unique finding of Dpm1-related pathways rescuing DPAGT1 inhibition, and reveal a novel interaction between fructose metabolism and ER stress.

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Genome-wide CRISPR Screen to Identify Genes that Suppress Transformation in the Presence of Endogenous KrasG12D

Scientific Reports ◽

10.1038/s41598-019-53572-w ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Jianguo Huang ◽

Mark Chen ◽

Eric S. Xu ◽

Lixia Luo ◽

Yan Ma ◽

...

Keyword(s):

Kinase Inhibitor ◽

Gene Mutations ◽

Soft Agar ◽

Reading Frame ◽

Immunodeficient Mice ◽

Genome Wide ◽

A Genome ◽

Crispr Screen

AbstractCooperating gene mutations are typically required to transform normal cells enabling growth in soft agar or in immunodeficient mice. For example, mutations in Kras and transformation-related protein 53 (Trp53) are known to transform a variety of mesenchymal and epithelial cells in vitro and in vivo. Identifying other genes that can cooperate with oncogenic Kras and substitute for Trp53 mutation has the potential to lead to new insights into mechanisms of carcinogenesis. Here, we applied a genome-wide CRISPR/Cas9 knockout screen in KrasG12D immortalized mouse embryonic fibroblasts (MEFs) to search for genes that when mutated cooperate with oncogenic Kras to induce transformation. We also tested if mutation of the identified candidate genes could cooperate with KrasG12D to generate primary sarcomas in mice. In addition to identifying the well-known tumor suppressor cyclin dependent kinase inhibitor 2A (Cdkn2a), whose alternative reading frame product p19 activates Trp53, we also identified other putative tumor suppressors, such as F-box/WD repeat-containing protein 7 (Fbxw7) and solute carrier family 9 member 3 (Slc9a3). Remarkably, the TCGA database indicates that both FBXW7 and SLC9A3 are commonly co-mutated with KRAS in human cancers. However, we found that only mutation of Trp53 or Cdkn2a, but not Fbxw7 or Slc9a3 can cooperate with KrasG12D to generate primary sarcomas in mice. These results show that mutations in oncogenic Kras and either Fbxw7 or Slc9a3 are sufficient for transformation in vitro, but not for in vivo sarcomagenesis.

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Genome-wide CRISPR screen identifies TMEM41B as a gene required for autophagosome formation

The Journal of Cell Biology ◽

10.1083/jcb.201804132 ◽

2018 ◽

Vol 217 (11) ◽

pp. 3817-3828 ◽

Cited By ~ 44

Author(s):

Keigo Morita ◽

Yutaro Hama ◽

Tamaki Izume ◽

Norito Tamura ◽

Toshihide Ueno ◽

...

Keyword(s):

Membrane Protein ◽

Degradation Process ◽

Autophagic Flux ◽

Autophagosome Formation ◽

Early Step ◽

Genome Wide ◽

A Genome ◽

Crispr Screen

Macroautophagy is an intracellular degradation process that requires multiple autophagy-related (ATG) genes. In this study, we performed a genome-wide screen using the autophagic flux reporter GFP-LC3-RFP and identified TMEM41B as a novel ATG gene. TMEM41B is a multispanning membrane protein localized in the endoplasmic reticulum (ER). It has a conserved domain also found in vacuole membrane protein 1 (VMP1), another ER multispanning membrane protein essential for autophagy, yeast Tvp38, and the bacterial DedA family of putative half-transporters. Deletion of TMEM41B blocked the formation of autophagosomes at an early step, causing accumulation of ATG proteins and small vesicles but not elongating autophagosome-like structures. Furthermore, lipid droplets accumulated in TMEM41B-knockout (KO) cells. The phenotype of TMEM41B-KO cells resembled those of VMP1-KO cells. Indeed, TMEM41B and VMP1 formed a complex in vivo and in vitro, and overexpression of VMP1 restored autophagic flux in TMEM41B-KO cells. These results suggest that TMEM41B and VMP1 function together at an early step of autophagosome formation.

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IPO11 mediates βcatenin nuclear import in a subset of colorectal cancers

The Journal of Cell Biology ◽

10.1083/jcb.201903017 ◽

2019 ◽

Vol 219 (2) ◽

Cited By ~ 4

Author(s):

Monika Mis ◽

Siobhan O’Brien ◽

Zachary Steinhart ◽

Sichun Lin ◽

Traver Hart ◽

...

Keyword(s):

Nuclear Import ◽

Gene Activation ◽

Protein Stabilization ◽

Nuclear Accumulation ◽

Colorectal Cancers ◽

Protein Levels ◽

Genome Wide ◽

A Genome ◽

Crispr Screen ◽

Context Specific

Activation of Wnt signaling entails βcatenin protein stabilization and translocation to the nucleus to regulate context-specific transcriptional programs. The majority of colorectal cancers (CRCs) initiate following APC mutations, resulting in Wnt ligand—independent stabilization and nuclear accumulation of βcatenin. The mechanisms underlying βcatenin nucleocytoplasmic shuttling remain incompletely defined. Using a novel, positive selection, functional genomic strategy, DEADPOOL, we performed a genome-wide CRISPR screen and identified IPO11 as a required factor for βcatenin-mediated transcription in APC mutant CRC cells. IPO11 (Importin-11) is a nuclear import protein that shuttles cargo from the cytoplasm to the nucleus. IPO11−/− cells exhibit reduced nuclear βcatenin protein levels and decreased βcatenin target gene activation, suggesting IPO11 facilitates βcatenin nuclear import. IPO11 knockout decreased colony formation of CRC cell lines and decreased proliferation of patient-derived CRC organoids. Our findings uncover a novel nuclear import mechanism for βcatenin in cells with high Wnt activity.

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Genome-wide screens identify Toxoplasma gondii determinants of parasite fitness in IFNγ-activated murine macrophages

Nature Communications ◽

10.1038/s41467-020-18991-8 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Yifan Wang ◽

Lamba Omar Sangaré ◽

Tatiana C. Paredes-Santos ◽

Musa A. Hassan ◽

Shruthi Krishnamurthy ◽

...

Keyword(s):

Dense Granule ◽

Cell Type ◽

Murine Macrophages ◽

Parasite Fitness ◽

Early Immune Response ◽

Host Cytoplasm ◽

Genome Wide ◽

A Genome ◽

Crispr Screen

Abstract Macrophages play an essential role in the early immune response against Toxoplasma and are the cell type preferentially infected by the parasite in vivo. Interferon gamma (IFNγ) elicits a variety of anti-Toxoplasma activities in macrophages. Using a genome-wide CRISPR screen we identify 353 Toxoplasma genes that determine parasite fitness in naїve or IFNγ-activated murine macrophages, seven of which are further confirmed. We show that one of these genes encodes dense granule protein GRA45, which has a chaperone-like domain, is critical for correct localization of GRAs into the PVM and secretion of GRA effectors into the host cytoplasm. Parasites lacking GRA45 are more susceptible to IFNγ-mediated growth inhibition and have reduced virulence in mice. Together, we identify and characterize an important chaperone-like GRA in Toxoplasma and provide a resource for the community to further explore the function of Toxoplasma genes that determine fitness in IFNγ-activated macrophages.

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A genome-wide, in vivo dropout CRISPR screen in acute myeloid leukemia

Experimental Hematology ◽

10.1016/j.exphem.2017.06.169 ◽

2017 ◽

Vol 53 ◽

pp. S78-S79

Author(s):

Francois Mercier ◽

Jiantao Shi ◽

David Sykes ◽

Toshihiko Oki ◽

Elisabeth Miller ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Genome Wide ◽

A Genome ◽

Crispr Screen ◽

Acute Myeloid

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A genome-wide CRISPR screen identifies UFMylation and TRAMP-like complexes as host factors required for hepatitis A virus infection

Cell Reports ◽

10.1016/j.celrep.2021.108859 ◽

2021 ◽

Vol 34 (11) ◽

pp. 108859

Author(s):

Jessie Kulsuptrakul ◽

Ruofan Wang ◽

Nathan L. Meyers ◽

Melanie Ott ◽

Andreas S. Puschnik

Keyword(s):

Virus Infection ◽

Hepatitis A ◽

Hepatitis A Virus ◽

Host Factors ◽

Genome Wide ◽

A Genome ◽

Crispr Screen

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Co-Overexpression of TWIST1-CSF1 Is a Common Event in Metastatic Oral Cancer and Drives Biologically Aggressive Phenotype

Cancers ◽

10.3390/cancers13010153 ◽

2021 ◽

Vol 13 (1) ◽

pp. 153

Author(s):

Sabrina Daniela da Silva ◽

Fabio Albuquerque Marchi ◽

Jie Su ◽

Long Yang ◽

Ludmila Valverde ◽

...

Keyword(s):

Epithelial Mesenchymal Transition ◽

Inflammatory Cells ◽

Rank Test ◽

Mesenchymal Transition ◽

Genome Wide ◽

A Genome ◽

Enhanced Expression ◽

Advanced Stages

Invasive oral squamous cell carcinoma (OSCC) is often ulcerated and heavily infiltrated by pro-inflammatory cells. We conducted a genome-wide profiling of tissues from OSCC patients (early versus advanced stages) with 10 years follow-up. Co-amplification and co-overexpression of TWIST1, a transcriptional activator of epithelial-mesenchymal-transition (EMT), and colony-stimulating factor-1 (CSF1), a major chemotactic agent for tumor-associated macrophages (TAMs), were observed in metastatic OSCC cases. The overexpression of these markers strongly predicted poor patient survival (log-rank test, p = 0.0035 and p = 0.0219). Protein analysis confirmed the enhanced expression of TWIST1 and CSF1 in metastatic tissues. In preclinical models using OSCC cell lines, macrophages, and an in vivo matrigel plug assay, we demonstrated that TWIST1 gene overexpression induces the activation of CSF1 while TWIST1 gene silencing down-regulates CSF1 preventing OSCC invasion. Furthermore, excessive macrophage activation and polarization was observed in co-culture system involving OSCC cells overexpressing TWIST1. In summary, this study provides insight into the cooperation between TWIST1 transcription factor and CSF1 to promote OSCC invasiveness and opens up the potential therapeutic utility of currently developed antibodies and small molecules targeting cancer-associated macrophages.

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