Líffræðifélag Íslands - biologia.is
Líffræðiráðstefnan 2023
Höfundar / Authors: Hlynur Magnússon(1), Abbi E. Smith(1), Sara Sigurbjörnsdóttir (1)
Starfsvettvangur / Affiliations: 1. Háskóli Íslands
Kynnir / Presenter: Hlynur Magnússon
Osteoarthritis (OA) is a widespread joint disease influenced by various genetic and environmental factors. A missense mutation in the human Smoothened (SMO) gene (R173C, corresponding to H177C in mouse) has been associated with increased susceptibility to OA. SMO is a key player in the hedgehog signaling pathway and the SMOR173C mutation is located close to the cholesterol binding region of SMO and is therefore thought to affect cholesterol binding of SMO, a crucial event in hedgehog signaling activation. Our lab has previously utilized an SMO overexpression system to study the effect the SMOR173C mutation has on OA but that may not accurately represent biological processes that occur at endogenous levels. To study the mutation at endogenous levels we have adapted a recently published approach to use CRISPR/Cas9 to generate an endogenously expressing SMOH177C mouse chondrogenic cell line that will be used to study its effect on OA. By harnessing homologous directed repair (HDR) and implementing a triple transfection of CRISPR/Cas9 components as well as synchronizing the cells using nocodazole, we significantly enhanced HDR efficiency. Using this method we successfully established a cell line harboring the OA related SMO missense mutation. Our optimized technique holds promise for advancing gene editing efficiency, and its application in generating a missense mutation shown to increase suseptibility to OA will enhance our understanding of OA pathology.