GM2 mediated YAP/TAZ regulation: elucidation of critical involvement of the master regulator LATS kinases

Abstract

Hippo signaling is an evolutionarily conserved regulator of tissue growth and cell fate. LATS1/2 is the upstream kinase of the Hippo signaling pathway which regulates phosphorylation and dephosphorylation of YAP/TAZ to modulate nuclear-cytoplasmic shuttling. To define the role of GM2 in regulation of Hippo pathway, here we aimed to generate LATS2 knockout cell line. CRISPR-Cas9 genome engineering technology has been applied to achieve this goal. Here, we have designed and constructed CRISPR-Cas9 plasmid against coding sequence of LATS2. The sequence verified CRISPR construct against human LATS2 were then transfected into HeLa cells to assess the functionality and to generate LATS2 knockout Hela cell Line. Further, LATS2 CRISPR construct transfected Hela cells were selected and clonal selection strategy was undertaken to select, expand and clonally purify LATS2 knockout Hela cells. Here, we have shown that CRISPR-Cas9 construct against LATS2 is functional and we were also able to generate LATS2 knockout Hela cell line. This LATS2 knockout cell will help us to understand the role GM2 in regulation of Hippo signaling in future studies.