Author(s): Kolton Obray, Spencer Perry
Mentor(s): Daniel Clark
Institution Weber
Enterovirus 71 (EV71) and herpes simplex 1 (HSV-1) are viruses that cause skin lesions in humans. EV71 causes hand foot and mouth disease (HFMD) and primarily affects young children. HSV-1 is a lifelong infection, causing genital herpes and cold sores, which affects 50 to 80 percent of US adults. CRISPR was used to edit the human genome in cultured cells (HEK293 and HeLa) to decrease the infectivity of these two viruses by deleting their receptors. To delete these virus receptors, a guide RNA (gRNA) was designed for each receptor (ANXA2, SCARB2, and SELPLG for EV71, and Nectin-1 and HVEM for HSV-1). Plasmids that express each gRNA and the CRISPR cutting enzyme, Cas9, were transfected into human cells using the base plasmid All_in_one_CRISPR. This plasmid contains a dsRed fluorescent protein and a G418-selectable marker for the selection of transfected cells, which were then confirmed as knockouts by Sanger sequencing. Cells were then infected and compared to wild-type for viability or viral-induced apoptosis. Due to the multiple combinations of receptors that viruses use, receptors that are most critical for attachment and entry into human cells can be determined. Through viability testing it was found that HeLa cells infected with HSV-1 were roughly fifty percent viable, in both HVEM-knockout and wild-type HeLa cells. However, when the Nectin-1 receptor was knocked out in HeLa cells, the cells remained fully viable, after exposure to HSV-1. By better understanding the interactions between virus and cell receptors, this research will lead to the development of targeted antiviral drugs that block viral attachment.