TGFB1 and osteosarcoma: Using recently developed super-efficient CRISP/Cas9 system in a doxycycline-inducible osteosarcoma U2OS Stably Expressing Cas9-Cells (SEC-C),32 we co-transfected a vector encoding sgRNA targeting the start codon region of the PAI-1 gene (sgPAI-1) together with the TGFβ reporter donor vector containing two homologous regions (~500nts) with the PAI-1 gene, one upstream (L-Arm) and one downstream (R-Arm) of the luciferase and GFP reporter cassette, to allow for homologous recombination (Fig. 1a).