A CRISPR Activation Screen Identifies a Pan-avian Influenza Virus Inhibitory Host Factor

Heaton, Brook E.; Kennedy, Edward M.; Dumm, Rebekah E.; Harding, Alfred T.; Sacco, Matthew T.; Sachs, David; Heaton, Nicholas S.

doi:10.1016/j.celrep.2017.07.060

Cited by 115 publications

(105 citation statements)

References 43 publications

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“…Functional genomic and genetic screens, based on siRNA, insertional mutagenesis and, more recently, CRISPR/Cas9, have been extremely valuable in identifying host factors affecting viral infectivity and replication, in particular regarding entry factors 16,17,[36][37][38] . However, the role of metabolic genes and transporters as host genes affecting the viral life cycle and host cell survival upon infection remain relatively understudied.…”

Section: Discussionmentioning

confidence: 99%

The transporters SLC35A1 and SLC30A1 play opposite roles in cell survival upon VSV virus infection

Moskovskich

Goldmann

Kartnig

et al. 2019

Preprint

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Host factor requirements for different classes of viruses are still to be fully unraveled. Replication of the viral genome and synthesis of viral proteins inside the human host cell are associated with altered, often enhanced, cellular metabolism and increased demand for nutrients as well as specific metabolites. With more than 400 members listed to date in humans, solute carriers (SLCs) represent the largest family of transmembrane proteins dedicated to the transport of ions and small molecules such as amino acids, sugars and nucleotides. Consistent with their impact on cellular metabolism, several SLCs have been implicated as host factors affecting the viral life cycle and the cell response to infection. In this study, we aimed at characterizing the role of host SLCs in cell survival upon viral infection by performing unbiased genetic screens using a focused CRISPR knockout library. Genetic screens with the cytolytic vesicular stomatitis virus (VSV) showed that loss of two SLCs genes, encoding the sialic acid transporter SLC35A1/CST and the zinc transporter SLC30A1/ZnT1, affected cell survival upon infection. Further characterization of these genes pointed to a role of both transporters in the apoptotic response induced by VSV, offering new insights into the cellular response to oncolytic virus infections.

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Section: Discussionmentioning

confidence: 99%

The transporters SLC35A1 and SLC30A1 play opposite roles in cell survival upon VSV virus infection

Moskovskich

Goldmann

Kartnig

et al. 2019

Preprint

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“…Recent advances in CRISPR-Cas9 have allowed highly efficient generation of knockout cells with marked phenotypes for the identification of virus dependency factors with very few false positive hits. CRISPR-Cas9 screens using multiplexed pools of sgRNAs that cover the entire human genome have proved to be a powerful technologic advance and have been successfully used to identify receptors and other cellular dependency factors for several viruses, including influenza A virus, Zika virus, West Nile virus, dengue virus and hepatitis C virus (53, 54, 59-61), reviewed in (55).…”

Section: Discussionmentioning

confidence: 99%

Identification of A Disintegrin and Metalloproteinase 9 domain (ADAM9) required in the early stages of encephalomyocarditis virus infection

King

et al. 2018

Preprint

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word count: 165 words 21 Main text word count: 5,097 words 22 Abstract 23 Encephalomyocarditis virus (EMCV) is a picornavirus that produces lytic 24 infections in murine and human cells. Employing a genome-wide CRISPR-Cas9 25 knockout screen to find host factors required for EMCV infection, we identified a role for 26 ADAM9 in EMCV infection. CRISPR-mediated deletion of ADAM9 in multiple human cell 27 lines rendered the cells highly resistant to EMCV infection and cell death. Primary 28 fibroblasts from ADAM9 KO mice were also strongly resistant to EMCV infection and cell 29 death. In contrast, ADAM9 KO and WT cells were equally susceptible to infection with 30 other viruses, including the picornavirus Coxsackie virus B. ADAM9 KO cells failed to 31 produce viral progeny when incubated with EMCV. However, bypassing EMCV entry into 32 cells through delivery of viral RNA directly to the cytosol yielded infectious EMCV virions 33 from ADAM9 KO cells, suggesting that ADAM9 is not required for EMCV replication 34 post-entry. These findings establish that ADAM9 is required for the early stage of EMCV 35 infection, likely for virus entry or viral genome delivery to the cytosol. 36 Importance 37 Viral myocarditis is a leading cause of death in the U.S., contributing to 38 numerous unexplained deaths in people ≤ 35 years old. Enteroviruses contribute to 39 many cases of human myocarditis. Encephalomyocarditis virus (EMCV) infection causes 40 viral myocarditis in rodent models but its receptor requirements have not been fully 41 identified. CRISPR-Cas9 screens can identify host dependency factors essential for 42 EMCV infection and enhance our understanding of key events that follow viral infection, 43 potentially leading to new strategies for preventing viral myocarditis. Using a CRISPR-44 Cas9 screen, we identified A Disintegrin and Metalloproteinase 9 Domain (ADAM9) as a 45 major factor required for the early stages of EMCV infection in both human and murine 46 infection. 47 Results 90 CRISPR-Cas9 Screening Identifies EMCV Dependency Factors (EDFs) 91EMCV infection is rapidly lytic in human and murine cells (51-54). We took advantage of 92 the high lytic potential of EMCV and the power of CRISPR genetic screening (53, 55) to 93 discover virus-host interaction genes that mediated virus infection and, thus, rendered 94 the cells susceptible to EMCV-induced cell death. HeLa cells stably expressing Cas9 95 were used for screening (53, 55). In initial optimization experiments, we determined that 96 HeLa cells were killed by EMCV within 24 h of infection at a multiplicity of infection (MOI) 97 ≥ 0.1. The rapid lysis of HeLa cells with EMCV infection allowed us to screen for EDFs 98 using pooled single-guide RNAs (sgRNAs) since we could identify such mutant cells by 99 their resistance to EMCV-induced cell death, i.e., these mutants would no longer be 100 susceptible to EMCV infection and would survive EMCV challenge. 101We screened for EDFs using CRISPR-Cas9 pooled human gene screen (Fig. 1). 102 H1-HeLa cells which stably exp...

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“…To do this, we transfected a pool of 6 crRNAs targeting a given IAV genome segment. Two days after crRNA transfection, the Cas13d A549 cells were challenged with PR8-mNeon, a strain of H1N1 IAV (A/Puerto Rico/8/1934) engineered to express the mNeonGreen gene, a fluorescent reporter protein (hereafter referred to as PR8-mNeon) at an MOI of 2.5 or 5.0 [26][27][28] . At approximately 18 hours post-challenge, the cells were analyzed for IAV infection through flow cytometry and microscopy.…”

Section: Crispr Pac-man Is Able To Inhibit Iav Infection In Human Lunmentioning

confidence: 99%

Development of CRISPR as a prophylactic strategy to combat novel coronavirus and influenza

Abbott

Dhamdhere

Liu

et al. 2020

Preprint

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The outbreak of the coronavirus disease 2019 , caused by the Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), has infected more than 100,000 people worldwide with over 3,000 deaths since December 2019. There is no cure for COVID-19 and the vaccine development is estimated to require 12-18 months. Here we demonstrate a CRISPR-Cas13-based strategy, PAC-MAN (Prophylactic Antiviral CRISPR in huMAN cells), for viral inhibition that can effectively degrade SARS-CoV-2 sequences and live influenza A virus (IAV) genome in human lung epithelial cells. We designed and screened a group of CRISPR RNAs (crRNAs) targeting conserved viral regions and identified functional crRNAs for cleaving SARS-CoV-2. The approach is effective in reducing respiratory cell viral replication for H1N1 IAV. Our bioinformatic analysis showed a group of only six crRNAs can target more than 90% of all coronaviruses. The PAC-MAN approach is potentially a rapidly implementable pan-coronavirus strategy to deal with emerging pandemic strains.

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A CRISPR Activation Screen Identifies a Pan-avian Influenza Virus Inhibitory Host Factor

Cited by 115 publications

References 43 publications

The transporters SLC35A1 and SLC30A1 play opposite roles in cell survival upon VSV virus infection

The transporters SLC35A1 and SLC30A1 play opposite roles in cell survival upon VSV virus infection

Identification of A Disintegrin and Metalloproteinase 9 domain (ADAM9) required in the early stages of encephalomyocarditis virus infection

Development of CRISPR as a prophylactic strategy to combat novel coronavirus and influenza

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