Poster #092, University of California, San Francisco
CRISPR engineering of microtubule regulatory protein in human induced pluripotent stem cells
Mentors: Yasmeen Ibrahim and Torsten Wittmann,PhD
Microtubules (MT) are structural components of the cell, providing structure and stability through a dynamic cytoskeletal network. Microtubule-associated proteins (MAPs) play a critical role in neuronal migration, the process by which neurons move to their correct positions in a developing brain. One example is Doublecortin (DCX), which stabilizes MTs. Another MAP is Doublecortin-like kinase 1 (DCLK1), which contains a domain that functions similarly to DCX by also promoting MT stabilization. Studies in mice have shown that mutations in both DCX and DCLK1 result in brain structure malformation and tumors. However, it is unknown if the same occurs in human models. To investigate this, we used human induced pluripotent stem cells (hiPSCs) in which DCX and DCLK-1 were endogenously tagged with mStayGold (mSG), a green fluorescent protein (GFP), using CRISPR/Cas9 genome editing. Because neither protein is expressed in hiPSCs, successful mSG integration was confirmed by PCR rather than fluorescence. Gel electrophoresis will show successful integration of mSG; a band of 563 bases will indicate the absence of the mSG sequence, while a 1178 band will confirm its presence. To study the effects of DCX and DCLK1 absence, the lack of mSG signal will indicate the successful removal of such proteins.