Authors: Rylan Schmanski, Mason Masters, Emilee Snow, Alexandria Offringa, Paola Robles, Spencer Perry, Joshua Mackley, Alexander Beagley, Rainey Hughes. Mentors: Daniel Clark. Insitution: Weber State University. 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. We used CRISPR 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 using the Broad Institute gRNA design tool (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 for viral-induced apoptosis. Because viruses use combinations of receptors, the end goal is to determine which receptors are most critical for attachment and entry into human cells. This will lead to targeted antiviral drugs to block interactions with those receptors.
