Using CRISPR-Cas9 Applications for ACE2 Knockout in Liver Epithelial Stem Cells and Impact on SARS-CoV-2



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The emerging Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) poses a major threat to public health. COVID-19 is a viral respiratory illness caused by SARS-CoV-2 and can be contracted between individuals who are in close contact with one another or by touching a contaminated object. SARS-CoV-2 entry depends on the host cell factors, ACE2 and TMPRSS2. Angiotensin I Converting Enzyme 2 (ACE2) is a functional receptor for the spike glycoprotein SARS-CoV-2 and TMPRSS2 is a transmembrane protease that serves as a primer for SARS-CoV-2 entry into the cell. ACE2 is expressed in the human airway epithelium, gastrointestinal cells, and some organs of the digestive system such as the liver. My objective is to knock out ACE2 in liver epithelial stem cells using CRISPR Cas9 technology as a means for preventing the entry of SARS-CoV-2. This process involves using chemical-based transfection in order to insert plasmids with the target gRNA and Cas9 enzyme as well as a GFP reporter that would serve as a marker for cells that have been edited. After transfection, positive selection with GFP reporting signal will be done and it will be followed by DNA analysis through sequencing. From this, we can infect the edited cells with SARS-CoV-2 and assess the effectiveness of the gene knockout on the prevention of COVID-19.