In Vitro Inhibition of Influenza Virus Using CRISPR/Cas13a in Chicken Cells
Advances in the field of CRISPR/Cas systems are expanding our ability to modulate cellular genomes and transcriptomes precisely and efficiently. Here, we assessed the Cas13a-mediated targeted disruption of RNA in chicken fibroblast DF1 cells. First, we developed a Tol2 transposon vector carrying the...
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MDPI AG
2021-06-01
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| Online Access: | https://www.mdpi.com/2409-9279/4/2/40 |
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| author | Arjun Challagulla Karel A. Schat Timothy J. Doran |
| author_facet | Arjun Challagulla Karel A. Schat Timothy J. Doran |
| author_sort | Arjun Challagulla |
| collection | DOAJ |
| description | Advances in the field of CRISPR/Cas systems are expanding our ability to modulate cellular genomes and transcriptomes precisely and efficiently. Here, we assessed the Cas13a-mediated targeted disruption of RNA in chicken fibroblast DF1 cells. First, we developed a Tol2 transposon vector carrying the Cas13a-msGFP-NLS (pT-Cas13a) transgene, followed by a stable insertion of the Cas13a transgene into the genome of DF1 cells to generate stable DF1-Cas13a cells. To assess the Cas13a-mediated functional knockdown, DF1-Cas13a cells were transfected with the combination of a plasmid encoding DsRed coding sequence (pDsRed) and DsRed-specific crRNA (crRNA-DsRed) or non-specific crRNA (crRNA-NS). Fluorescence-activated cell sorting (FACS) and a microscopy analysis showed reduced levels of DsRed expression in cells transfected with crRNA-DsRed but not in crRNA-NS, confirming a sequence-specific Cas13a mediated mRNA knockdown. Next, we designed four crRNAs (crRNA-IAV) against the PB1, NP and M genes of influenza A virus (IAV) and cloned in tandem to express from a single vector. DF1-Cas13a cells were transfected with plasmids encoding the crRNA-IAV or crRNA-NS, followed by infection with WSN or PR8 IAV. DF1 cells transfected with crRNA-IAV showed reduced levels of viral titers compared to cells transfected with crRNA-NS. These results demonstrate the potential of Cas13a as an antiviral strategy against highly pathogenic strains of IAV in chickens. |
| first_indexed | 2024-03-10T10:37:11Z |
| format | Article |
| id | doaj.art-790165d038ec4ba6ae0bbfe82dbc3a41 |
| institution | Directory Open Access Journal |
| issn | 2409-9279 |
| language | English |
| last_indexed | 2024-03-10T10:37:11Z |
| publishDate | 2021-06-01 |
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| series | Methods and Protocols |
| spelling | doaj.art-790165d038ec4ba6ae0bbfe82dbc3a412023-11-21T23:15:18ZengMDPI AGMethods and Protocols2409-92792021-06-01424010.3390/mps4020040In Vitro Inhibition of Influenza Virus Using CRISPR/Cas13a in Chicken CellsArjun Challagulla0Karel A. Schat1Timothy J. Doran2CSIRO Health and Biosecurity, Australian Centre for Disease Preparedness, Geelong 3220, AustraliaDepartment of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USACSIRO Health and Biosecurity, Australian Centre for Disease Preparedness, Geelong 3220, AustraliaAdvances in the field of CRISPR/Cas systems are expanding our ability to modulate cellular genomes and transcriptomes precisely and efficiently. Here, we assessed the Cas13a-mediated targeted disruption of RNA in chicken fibroblast DF1 cells. First, we developed a Tol2 transposon vector carrying the Cas13a-msGFP-NLS (pT-Cas13a) transgene, followed by a stable insertion of the Cas13a transgene into the genome of DF1 cells to generate stable DF1-Cas13a cells. To assess the Cas13a-mediated functional knockdown, DF1-Cas13a cells were transfected with the combination of a plasmid encoding DsRed coding sequence (pDsRed) and DsRed-specific crRNA (crRNA-DsRed) or non-specific crRNA (crRNA-NS). Fluorescence-activated cell sorting (FACS) and a microscopy analysis showed reduced levels of DsRed expression in cells transfected with crRNA-DsRed but not in crRNA-NS, confirming a sequence-specific Cas13a mediated mRNA knockdown. Next, we designed four crRNAs (crRNA-IAV) against the PB1, NP and M genes of influenza A virus (IAV) and cloned in tandem to express from a single vector. DF1-Cas13a cells were transfected with plasmids encoding the crRNA-IAV or crRNA-NS, followed by infection with WSN or PR8 IAV. DF1 cells transfected with crRNA-IAV showed reduced levels of viral titers compared to cells transfected with crRNA-NS. These results demonstrate the potential of Cas13a as an antiviral strategy against highly pathogenic strains of IAV in chickens.https://www.mdpi.com/2409-9279/4/2/40chickenCRISPR/Cas13acrRNAinfluenza A virus (IAV) |
| spellingShingle | Arjun Challagulla Karel A. Schat Timothy J. Doran In Vitro Inhibition of Influenza Virus Using CRISPR/Cas13a in Chicken Cells Methods and Protocols chicken CRISPR/Cas13a crRNA influenza A virus (IAV) |
| title | In Vitro Inhibition of Influenza Virus Using CRISPR/Cas13a in Chicken Cells |
| title_full | In Vitro Inhibition of Influenza Virus Using CRISPR/Cas13a in Chicken Cells |
| title_fullStr | In Vitro Inhibition of Influenza Virus Using CRISPR/Cas13a in Chicken Cells |
| title_full_unstemmed | In Vitro Inhibition of Influenza Virus Using CRISPR/Cas13a in Chicken Cells |
| title_short | In Vitro Inhibition of Influenza Virus Using CRISPR/Cas13a in Chicken Cells |
| title_sort | in vitro inhibition of influenza virus using crispr cas13a in chicken cells |
| topic | chicken CRISPR/Cas13a crRNA influenza A virus (IAV) |
| url | https://www.mdpi.com/2409-9279/4/2/40 |
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