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CRISPR/CAS9 Knockout of IFNα Receptor Mediated Antiviral Response in PBS-12SF Cell Line
Noah Lubben, 4th-year |
Abstract:
Viruses such influenza viruses and coronaviruses are some of the most widely spread infectious diseases in humans and animals. Many such diseases can be prevented through vaccination. Large-scale vaccination requires generation of massive quantities of vaccines both efficiently and rapidly. Cell lines offer a means to quickly generate large quantities of virus safely and provide an adaptable method for various viruses. Our lab has developed the PBS-12SF cell line that supports replication of many viruses, including avian and human influenza viruses. Viral infection of cells triggers the interferon (IFN) response carried out by type I IFNs via the IFN cell surface receptor and eliciting an antiviral response. In a previous demonstration project shRNA knock down of INF receptor (IFNAR1) mRNA reduced the IFN response, enhancing viral production in PBS-12SF cells. Currently, we are using CRISPR/CAS9 knockout of IFNAR1 to permanently modify PBS-12SF cells, crippling the antiviral response and enhancing viral production of influenza and other viruses, including adenovirus vectors used in some COVID-19 vaccines. To accomplish this, we first developed methods for detecting IFNAR1 protein on PBS-12SF cells. Next, we used a green fluorescent protein (GFP) expression plasmid to optimize transfection conditions, assessed by flow cytometry. We then co-transfected PBS-12SF cells with GFP plasmid and complexes of CRISPR guide RNAs+Cas9. GFP expressing cells were sorted using the Influx HT FACS at the MSU Flow Core Facility. We are currently growing both single cell clones (>1000) and larger scale cultures of sorted GFP+ PBS-12SF cells for testing of successful IFNRA1 knockout.