CRISPR genetic screens to discover host–virus interactions

McDougall, William M.; Perreira, Jill M.; Reynolds, Erin C; Brass, Abraham L.

doi:10.1016/j.coviro.2018.03.007

Cited by 25 publications

(15 citation statements)

References 48 publications

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“…EMCV infection is rapidly lytic in human and murine cells (51–54). We took advantage of the high lytic potential of EMCV and the power of CRISPR genetic screening (53, 55) to discover virus-host interaction genes that mediated virus infection and, thus, rendered the cells susceptible to EMCV-induced cell death. HeLa cells stably expressing Cas9 were used for screening (53, 55).…”

Section: Resultsmentioning

confidence: 99%

“…We took advantage of the high lytic potential of EMCV and the power of CRISPR genetic screening (53, 55) to discover virus-host interaction genes that mediated virus infection and, thus, rendered the cells susceptible to EMCV-induced cell death. HeLa cells stably expressing Cas9 were used for screening (53, 55). In initial optimization experiments, we determined that HeLa cells were killed by EMCV within 24 h of infection at a multiplicity of infection (MOI) of ≥0.1.…”

Section: Resultsmentioning

confidence: 99%

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A Disintegrin and Metalloproteinase 9 Domain (ADAM9) Is a Major Susceptibility Factor in the Early Stages of Encephalomyocarditis Virus Infection

Bazzone

King

MacKay

et al. 2019

mBio

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Encephalomyocarditis virus (EMCV) is a picornavirus that produces lytic infections in murine and human cells. Employing a genome-wide CRISPR-Cas9 knockout screen to find host factors required for EMCV infection, we identified a role for ADAM9 in EMCV infection. CRISPR-mediated deletion of ADAM9 in multiple human cell lines rendered the cells highly resistant to EMCV infection and cell death. Primary fibroblasts from ADAM9 KO mice were also strongly resistant to EMCV infection and cell death. In contrast, ADAM9 KO and WT cells were equally susceptible to infection with other viruses, including the picornavirus Coxsackie virus B. ADAM9 KO cells failed to produce viral progeny when incubated with EMCV. However, bypassing EMCV entry into cells through delivery of viral RNA directly to the cytosol yielded infectious EMCV virions from ADAM9 KO cells, suggesting that ADAM9 is not required for EMCV replication post-entry. These findings establish that ADAM9 is required for the early stage of EMCV infection, likely for virus entry or viral genome delivery to the cytosol.IMPORTANCEViral myocarditis is a leading cause of death in the United States, contributing to numerous unexplained deaths in people ≤35 years old. Enteroviruses contribute to many cases of human myocarditis. Encephalomyocarditis virus (EMCV) infection causes viral myocarditis in rodent models, but its receptor requirements have not been fully identified. CRISPR-Cas9 screens can identify host dependency factors essential for EMCV infection and enhance our understanding of key events that follow viral infection, potentially leading to new strategies for preventing viral myocarditis. Using a CRISPR-Cas9 screen, we identifiedadisintegrinandmetalloproteinase 9 domain (ADAM9) as a major factor required for the early stages of EMCV infection in both human and murine infection.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

A Disintegrin and Metalloproteinase 9 Domain (ADAM9) Is a Major Susceptibility Factor in the Early Stages of Encephalomyocarditis Virus Infection

Bazzone

King

MacKay

et al. 2019

mBio

View full text Add to dashboard Cite

show abstract

“…EMCV infection is rapidly lytic in human and murine cells (51-54). We took advantage of the high lytic potential of EMCV and the power of CRISPR genetic screening (53, 55) to discover virus-host interaction genes that mediated virus infection and, thus, rendered the cells susceptible to EMCV-induced cell death. HeLa cells stably expressing Cas9 were used for screening (53, 55).…”

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

“…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%

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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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Discovery of Zika Virus Dependency and Restriction Factors Using Flow-Based Arrayed CRISPR Screening for Identification of Targets (FACS-IT)

McDougall

Kandpal

Perreira

et al. 2020

Methods in Molecular Biology

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CRISPR genetic screens to discover host–virus interactions

Cited by 25 publications

References 48 publications

A Disintegrin and Metalloproteinase 9 Domain (ADAM9) Is a Major Susceptibility Factor in the Early Stages of Encephalomyocarditis Virus Infection

A Disintegrin and Metalloproteinase 9 Domain (ADAM9) Is a Major Susceptibility Factor in the Early Stages of Encephalomyocarditis Virus Infection

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

Discovery of Zika Virus Dependency and Restriction Factors Using Flow-Based Arrayed CRISPR Screening for Identification of Targets (FACS-IT)

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