|
| Status |
Public on Jun 27, 2022 |
| Title |
Differential Whole Genome CRISPR Screening Strategy for SARS-CoV-2 Spike and VSV-G Mediated Pseudoviral Entry |
| Organisms |
Homo sapiens; synthetic construct |
| Experiment type |
Other
|
| Summary |
The coronavirus disease 2019 (COVID-19) has caused over 6 million deaths worldwide and disrupted the global economy. The causative agent for this disease, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes mild to lethal respiratory infections. Understanding the cellular host factors that promote and inhibit for SARS-CoV-2 infection is important for identifying virial countermeasures. Moreover, new methods are needed to be to identify host genes influencing specific steps of viral infections. Here, we developed a CRISPR whole genome screen against SARS-CoV-2 spike enveloped retro-pseudoviruses with a GFP reporter to specifically identify host genes that facilitate viral entry. By including two counter screen strategies, this approach can be used to distinguish host genes affecting the pseudoviral reporter from those unique to envelope-mediated entry. First, an alternate envelope, VSV-G allowed identification of shared genes associated with retro-transcription, integration and reporter expression. Second, a recently developed Cre-Gag fusion pseudovirus bypassed retro transcription and integration by directly activating a floxed GFP reporter. Our approach correctly identified SARS-CoV-2 and VSV-G receptors ACE2 and LDLR, respectively and distinguished genes associated with retroviral reporter expression from envelope mediated entry. Overall, this work provides a new strategy for screening genes influencing envelope mediated entry without the complexity of live-viral screens which are complicated with large numbers of genes associated with all aspects of viral pathogenesis and replication. This approach should be of use for identifying genes contributing to and inhibiting SARS-CoV-2 entry and provide a platform for the analysis of newly emerging viruses.
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| Overall design |
CRISPR-Cas9 whole genome screen against SARS-CoV-2 spike-mNeonGreen, VSV-G-mNeonGreen and VSV-G-Cre pseudovirus for identifying host factors
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| Contributor(s) |
Saddam Hossain M, Kerkvliet JG, Hoppe AD |
| Citation(s) |
37707319 |
| |
| Submission date |
Jun 27, 2022 |
| Last update date |
Oct 24, 2024 |
| Contact name |
Adam D. Hoppe |
| E-mail(s) |
adam.hoppe@sdstate.edu
|
| Phone |
6056955140
|
| Organization name |
South Dakota State University
|
| Department |
Chemistry and Biochemistry
|
| Lab |
Immunology
|
| Street address |
1055 CAMPANILE AVE BOX 2202, Avera Health and Science
|
| City |
Brookings |
| State/province |
South Dakota |
| ZIP/Postal code |
57007 |
| Country |
USA |
| |
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| Platforms (2) |
|
| Samples (11)
|
| GSM6267740 |
Brunello_ACE2 293T_Spike_mNeonGreen_Presort_S2 |
| GSM6267741 |
Brunello_ACE2 293T_Spike_mNeonGreen_Low_S3 |
| GSM6267742 |
Brunello_ACE2 293T_Spike_mNeonGreen_High_S4 |
| GSM6267743 |
Brunlleo_ACE2 293T_VSVG_mNeonGreen_Presort_S5 |
| GSM6267744 |
Brunlleo_ACE2 293T_VSVG_mNeonGreen_Low_S6 |
| GSM6267745 |
Brunello_ACE2 293T_VSVG_mNeonGreen_High_S7 |
| GSM6267746 |
Brunello_Input_S8_Spike_VSVG |
| GSM6267747 |
Brunello_ACE2 293T-GFP floxed_VSVG_Cre_Presort_S2 |
| GSM6267748 |
Brunello_ACE2 293T-GFP floxed_VSVG_Cre_Low-S3 |
| GSM6267749 |
Brunello_ACE2 293T-GFP floxed_VSVG_Cre_High_S4 |
| GSM6267750 |
Brunello_Input_S1_VSVG_Cre |
|
| Relations |
| BioProject |
PRJNA853268 |
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