Creating a Heparanase Knockout in Hepatocellular Carcinoma Using CRISPR

CSU Ballroom

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related mortality and contributes to almost 700,000 deaths per year [1,2]. This cancer has a high metastatic rate and a high resistance to chemotherapeutics, with only one FDA-approved drug currently available. All these factors contribute to the need for new strategies and therapeutics for patients diagnosed with this cancer [2,3]. Glypican-3 (GPC3) proteins are attached to the cell surface and have been shown to be abundant in HCC compared to healthy tissue [3]. The heparan sulfate chains on glypican 3 proteins can be cleaved from the cell surface by the enzyme heparanase. When heparanase releases these chains, it changes the GPC3 structure that influences a variety of growth factor interactions and can affect the survival and invasion of tumor cells [2,3,4]. Heparanaselevels are higher in metastatic tumor cells and it is a known tumor promoter in most cancers [3,4] Using CRISPR technology, we can target the heparanase loci and create a functional knockout in HCC cells - rendering the enzyme dysfunctional. I have designed a guide RNA (sgRNA) that directs the Cas9 nuclease to an early exon in the heparanase loci. Using this sgRNA, I created a complex consisting of the sgRNA and Cas9 nuclease that was then delivered into HCC cells. During this period, the sgRNA brings Cas9 to the heparanase loci and causes a double-stranded break in the DNA. When this break occurs, the cells will try to fix it through a DNA repair mechanism – often via the error-prone non-homologous end joining (NHEJ) that will likely result in an early stop codon. These cells are now only able to make truncated, dysfunctional heparanase enzymes. We can use these cells in future experiments to study heparanase’s role and compare them to cells with a functional enzyme to gain more insight as to how heparanase influences HCC progression.