Establishing CRISPR-Cas9 and BioID Workflows to Probe Molecular Signaling Pathways in Kidney Podocytes
Podocyte detachment is a significant contributor to kidney disease progression, involving changes in signaling proteins which control integrin activity. Integrins are a major component of focal adhesions, and their activity is negatively regulated by the adaptor proteins Dok1 and Dok2. We uncovered a protective effect of Dok1 and Dok2 loss during podocyte injury in vivo, and as such, the goal of this thesis was to implement two new in vitro methodologies to better understand the role of Dok proteins in podocytes. Herein, we outline the development of workflows suited to the generation of CRISPR-Cas9 knockout (KO) podocytes as well as stable expressing bait-miniTurbo podocyte cell lines for proximity-dependent biotin identification (BioID). Dok1/2 CRISPR KO podocytes revealed a modest cell adhesion phenotype as well as increased cell migration. Cellular component gene ontology analysis of Dok1 and Dok2 proximity interactors revealed association of Dok proteins to adhesive junctions.