scholarly journals A genome-wide CRISPR screen identifies interactors of the autophagy pathway as conserved coronavirus targets

PLoS Biology ◽  
2021 ◽  
Vol 19 (12) ◽  
pp. e3001490
Author(s):  
Annika Kratzel ◽  
Jenna N. Kelly ◽  
Philip V’kovski ◽  
Jasmine Portmann ◽  
Yannick Brüggemann ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Huh7 Cell ◽  
Host Factors ◽  
Genome Wide ◽  
A Genome ◽  
Dependent Inhibition ◽  
Crispr Screen ◽  
Human Coronaviruses ◽  
Approved Drugs ◽  
Huh7 Cell Line

Over the past 20 years, 3 highly pathogenic human coronaviruses (HCoVs) have emerged—Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and, most recently, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)—demonstrating that coronaviruses (CoVs) pose a serious threat to human health and highlighting the importance of developing effective therapies against them. Similar to other viruses, CoVs are dependent on host factors for their survival and replication. We hypothesized that evolutionarily distinct CoVs may exploit similar host factors and pathways to support their replication cycles. Herein, we conducted 2 independent genome-wide CRISPR/Cas-9 knockout (KO) screens to identify MERS-CoV and HCoV-229E host dependency factors (HDFs) required for HCoV replication in the human Huh7 cell line. Top scoring genes were further validated and assessed in the context of MERS-CoV and HCoV-229E infection as well as SARS-CoV and SARS-CoV-2 infection. Strikingly, we found that several autophagy-related genes, including TMEM41B, MINAR1, and the immunophilin FKBP8, were common host factors required for pan-CoV replication. Importantly, inhibition of the immunophilin protein family with the compounds cyclosporine A, and the nonimmunosuppressive derivative alisporivir, resulted in dose-dependent inhibition of CoV replication in primary human nasal epithelial cell cultures, which recapitulate the natural site of virus replication. Overall, we identified host factors that are crucial for CoV replication and demonstrated that these factors constitute potential targets for therapeutic intervention by clinically approved drugs.

scholarly journals A genome-wide CRISPR screen identifies interactors of the autophagy pathway as conserved coronavirus targets

2021 ◽  
Author(s):  
Annika Kratzel ◽  
Jenna N. Kelly ◽  
Yannick Brueggemann ◽  
Jasmine Portmann ◽  
Philip V’kovski ◽  
...  
Keyword(s):  
Host Factors ◽  
The Novel ◽  
Genome Wide ◽  
A Genome ◽  
Dependent Inhibition ◽  
Crispr Screen ◽  
Dose Dependent Inhibition ◽  
Autophagy Pathway ◽  
Approved Drugs ◽  
Dose Dependent

SummaryOver the past 20 years, the emergence of three highly pathogenic coronaviruses (CoV) – SARS-CoV, MERS-CoV, and most recently SARS-CoV-2 – has shown that CoVs pose a serious risk to human health and highlighted the importance of developing effective therapies against them. Similar to other viruses, CoVs are dependent on host factors for their survival and replication. We hypothesized that evolutionarily distinct CoVs may exploit similar host factors and pathways to support their replication cycle. Here, we conducted two independent genome-wide CRISPR/Cas9 knockout screens to identify pan-CoV host factors required for the replication of both endemic and emerging CoVs, including the novel CoV SARS-CoV-2. Strikingly, we found that several autophagy-related genes, including the immunophilin FKBP8, TMEM41B, and MINAR1, were common host factors required for CoV replication. Importantly, inhibition of the immunophilin family with the compounds Tacrolimus, Cyclosporin A, and the non-immunosuppressive derivative Alisporivir, resulted in dose-dependent inhibition of CoV replication in primary human nasal epithelial cell cultures that resemble the natural site of virus replication. Overall, we identified host factors that are crucial for CoV replication and demonstrate that these factors constitute potential targets for therapeutic intervention by clinically approved drugs.

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

Cell Reports ◽  
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

scholarly journals Genome-wide CRISPR screen identifies host dependency factors for influenza A virus infection

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Bo Li ◽  
Sara M. Clohisey ◽  
Bing Shao Chia ◽  
Bo Wang ◽  
Ang Cui ◽  
...  
Keyword(s):  
Virus Infection ◽  
Influenza A Virus ◽  
Influenza A ◽  
Meta Analysis ◽  
Host Factors ◽  
Validation Data ◽  
Genome Wide ◽  
A Genome ◽  
Crispr Screen ◽  
Influenza A Virus Infection

AbstractHost dependency factors that are required for influenza A virus infection may serve as therapeutic targets as the virus is less likely to bypass them under drug-mediated selection pressure. Previous attempts to identify host factors have produced largely divergent results, with few overlapping hits across different studies. Here, we perform a genome-wide CRISPR/Cas9 screen and devise a new approach, meta-analysis by information content (MAIC) to systematically combine our results with prior evidence for influenza host factors. MAIC out-performs other meta-analysis methods when using our CRISPR screen as validation data. We validate the host factors, WDR7, CCDC115 and TMEM199, demonstrating that these genes are essential for viral entry and regulation of V-type ATPase assembly. We also find that CMTR1, a human mRNA cap methyltransferase, is required for efficient viral cap snatching and regulation of a cell autonomous immune response, and provides synergistic protection with the influenza endonuclease inhibitor Xofluza.

scholarly journals A genome-wide CRISPR screen identifies host factors that regulate SARS-CoV-2 entry

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yunkai Zhu ◽  
Fei Feng ◽  
Gaowei Hu ◽  
Yuyan Wang ◽  
Yin Yu ◽  
...  
Keyword(s):  
Critical Role ◽  
Surface Expression ◽  
Host Factors ◽  
Spike Protein ◽  
Hamster Model ◽  
Angiotensin Converting Enzyme 2 ◽  
Genome Wide ◽  
A Genome ◽  
Public Health Challenges ◽  
Crispr Screen

AbstractThe global spread of SARS-CoV-2 is posing major public health challenges. One feature of SARS-CoV-2 spike protein is the insertion of multi-basic residues at the S1/S2 subunit cleavage site. Here, we find that the virus with intact spike (Sfull) preferentially enters cells via fusion at the plasma membrane, whereas a clone (Sdel) with deletion disrupting the multi-basic S1/S2 site utilizes an endosomal entry pathway. Using Sdel as model, we perform a genome-wide CRISPR screen and identify several endosomal entry-specific regulators. Experimental validation of hits from the CRISPR screen shows that host factors regulating the surface expression of angiotensin-converting enzyme 2 (ACE2) affect entry of Sfull virus. Animal-to-animal transmission with the Sdel virus is reduced compared to Sfull in the hamster model. These findings highlight the critical role of the S1/S2 boundary of SARS-CoV-2 spike protein in modulating virus entry and transmission and provide insights into entry of coronaviruses.

scholarly journals A Genome-wide CRISPR Screen Identifies CDC25A as a Determinant of Sensitivity to ATR Inhibitors

2016 ◽  
Vol 62 (2) ◽  
pp. 307-313 ◽  
Author(s):  
Sergio Ruiz ◽  
Cristina Mayor-Ruiz ◽  
Vanesa Lafarga ◽  
Matilde Murga ◽  
Maria Vega-Sendino ◽  
...  
Keyword(s):  
Genome Wide ◽  
A Genome ◽  
Crispr Screen

scholarly journals Functional CRISPR screening identifies the ufmylation pathway as a regulator of SQSTM1/p62

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Rowena DeJesus ◽  
Francesca Moretti ◽  
Gregory McAllister ◽  
Zuncai Wang ◽  
Phil Bergman ◽  
...  
Keyword(s):  
Adaptor Protein ◽  
Er Stress Response ◽  
Genome Wide ◽  
A Genome ◽  
Crispr Screen ◽  
Selection Step ◽  
A Cell ◽  
Cell Type Specific ◽  
Cellular Pathways ◽  
Mtor Signalling

SQSTM1 is an adaptor protein that integrates multiple cellular signaling pathways and whose expression is tightly regulated at the transcriptional and post-translational level. Here, we describe a forward genetic screening paradigm exploiting CRISPR-mediated genome editing coupled to a cell selection step by FACS to identify regulators of SQSTM1. Through systematic comparison of pooled libraries, we show that CRISPR is superior to RNAi in identifying known SQSTM1 modulators. A genome-wide CRISPR screen exposed MTOR signalling and the entire macroautophagy machinery as key regulators of SQSTM1 and identified several novel modulators including HNRNPM, SLC39A14, SRRD, PGK1 and the ufmylation cascade. We show that ufmylation regulates SQSTM1 by eliciting a cell type-specific ER stress response which induces SQSTM1 expression and results in its accumulation in the cytosol. This study validates pooled CRISPR screening as a powerful method to map the repertoire of cellular pathways that regulate the fate of an individual target protein.

scholarly journals Genome-wide CRISPR screen identifies regulators of MAPK and MTOR pathways mediating sorafenib resistance in acute myeloid leukemia

Haematologica ◽  
2020 ◽  
Author(s):  
Alisa Damnernsawad ◽  
Daniel Bottomly ◽  
Stephen E. Kurtz ◽  
Christopher A. Eide ◽  
Shannon K. McWeeney ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Ex Vivo ◽  
Mek Inhibitors ◽  
Genome Wide ◽  
A Genome ◽  
Flt3 Mutations ◽  
Flt3 Inhibitors ◽  
Crispr Screen ◽  
Acute Myeloid

Drug resistance impedes the long-term effect of targeted therapies in acute myeloid leukemia (AML), necessitating the identification of mechanisms underlying resistance. Approximately 25% of AML patients carry FLT3 mutations and develop post-treatment insensitivity to FLT3 inhibitors, including sorafenib. Using a genome-wide CRISPR screen, we identified LZTR1, NF1, TSC1 or TSC2, negative regulators of the MAPK and MTOR pathways, as mediators of sorafenib resistance. Analyses of ex vivo drug sensitivity assays in FLT3-ITD AML patient samples revealed lower expression of LZTR1, NF1, and TSC2 correlated with sorafenib sensitivity. Importantly, MAPK and/or MTOR complex1 (MTORC1) activity were upregulated in AML cells made resistant to several FLT3 inhibitors, including crenolanib, quizartinib, or sorafenib. These cells were sensitive to MEK inhibitors, and the combination of FLT3 and MEK inhibitors showed enhanced efficacy, suggesting its effectiveness in AML patients with FLT3 mutations and those with resistance to FLT3 inhibitors.

scholarly journals Yersinia pestis Targets the Host Endosome Recycling Pathway during the Biogenesis of the Yersinia-Containing Vacuole To Avoid Killing by Macrophages

mBio ◽  
2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Michael G. Connor ◽  
Amanda R. Pulsifer ◽  
Donghoon Chung ◽  
Eric C. Rouchka ◽  
Brian K. Ceresa ◽  
...  
Keyword(s):  
Yersinia Pestis ◽  
Host Factors ◽  
Endocytic Recycling ◽  
Recycling Endosome ◽  
Independent Manner ◽  
Genome Wide ◽  
Type Three Secretion System ◽  
A Genome ◽  
Recycling Pathway ◽  
Bacterial Replication

ABSTRACTYersinia pestishas evolved many strategies to evade the innate immune system. One of these strategies is the ability to survive within macrophages. Upon phagocytosis,Y. pestisprevents phagolysosome maturation and establishes a modified compartment termed theYersinia-containing vacuole (YCV).Y. pestisactively inhibits the acidification of this compartment, and eventually, the YCV transitions from a tight-fitting vacuole into a spacious replicative vacuole. The mechanisms to generate the YCV have not been defined. However, we hypothesized that YCV biogenesis requiresY. pestisinteractions with specific host factors to subvert normal vesicular trafficking. In order to identify these factors, we performed a genome-wide RNA interference (RNAi) screen to identify host factors required forY. pestissurvival in macrophages. This screen revealed that 71 host proteins are required for intracellular survival ofY. pestis. Of particular interest was the enrichment for genes involved in endosome recycling. Moreover, we demonstrated thatY. pestisactively recruits Rab4a and Rab11b to the YCV in a type three secretion system-independent manner, indicating remodeling of the YCV byY. pestisto resemble a recycling endosome. While recruitment of Rab4a was necessary to inhibit YCV acidification and lysosomal fusion early during infection, Rab11b appeared to contribute to later stages of YCV biogenesis. We also discovered thatY. pestisdisrupts global host endocytic recycling in macrophages, possibly through sequestration of Rab11b, and this process is required for bacterial replication. These data provide the first evidence thatY. pestistargets the host endocytic recycling pathway to avoid phagolysosomal maturation and generate the YCV.IMPORTANCEYersinia pestiscan infect and survive within macrophages. However, the mechanisms that the bacterium use to subvert killing by these phagocytes have not been defined. To provide a better understanding of these mechanisms, we used an RNAi approach to identify host factors required for intracellularY. pestissurvival. This approach revealed that the host endocytic recycling pathway is essential forY. pestisto avoid clearance by the macrophage. We further demonstrate thatY. pestisremodels the phagosome to resemble a recycling endosome, allowing the bacterium to avoid the normal phagolysosomal maturation pathway. Moreover, we show that infection withY. pestisdisrupts normal recycling in the macrophage and that disruption is required for bacterial replication. These findings provide the first evidence thatY. pestistargets the host endocytic recycling pathway in order to evade killing by macrophages.

scholarly journals Genome-wide CRISPR screen identifies ELP5 as a determinant of gemcitabine sensitivity in gallbladder cancer

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Sunwang Xu ◽  
Ming Zhan ◽  
Cen Jiang ◽  
Min He ◽  
Linhua Yang ◽  
...  
Keyword(s):  
Gallbladder Cancer ◽  
Locally Advanced ◽  
Internal Ribosomal Entry Site ◽  
Dependent Manner ◽  
Entry Site ◽  
P53 Expression ◽  
Elongator Complex ◽  
Genome Wide ◽  
A Genome ◽  
Crispr Screen

AbstractGemcitabine is the first-line treatment for locally advanced and metastatic gallbladder cancer (GBC), but poor gemcitabine response is universal. Here, we utilize a genome-wide CRISPR screen to identify that loss of ELP5 reduces the gemcitabine-induced apoptosis in GBC cells in a P53-dependent manner through the Elongator complex and other uridine 34 (U34) tRNA-modifying enzymes. Mechanistically, loss of ELP5 impairs the integrity and stability of the Elongator complex to abrogate wobble U34 tRNA modification, and directly impedes the wobble U34 modification-dependent translation of hnRNPQ mRNA, a validated P53 internal ribosomal entry site (IRES) trans-acting factor. Downregulated hnRNPQ is unable to drive P53 IRES-dependent translation, but rescuing a U34 modification-independent hnRNPQ mutant could restore P53 translation and gemcitabine sensitivity in ELP5-depleted GBC cells. GBC patients with lower ELP5, hnRNPQ, or P53 expression have poor survival outcomes after gemcitabine chemotherapy. These results indicate that the Elongator/hnRNPQ/P53 axis controls gemcitabine sensitivity in GBC cells.

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