Targeted inactivation of EWSR1 : : FLI1 gene in Ewing sarcoma via CRISPR/Cas9 driven by an Ewing-specific GGAA promoter.

We have recently demonstrated that genetic inactivation of EWSR1 : : FLI1 by CRISPR/Cas9, successfully blocks cell proliferation in a cell model of Ewing sarcoma. However, CRISPR/Cas9-mediated gene editing can exhibit off-target effects, and thus, precise regulation of Cas9 expression in target cells is essential to develop gene-editing strategies to inactivate EWSR1 : : FLI1 in Ewing sarcoma cells. In this study, we demonstrate that Cas9 can be specifically expressed in Ewing sarcoma cells when located downstream a promoter consisting of GGAA repeats and a consensus TATA box (GGAAprom). Under these conditions, Cas9 is selectively expressed in Ewing sarcoma cells that express EWSR1 : : FLI1 oncoproteins, but not in cells expressing wild-type FLI1. Consequently, Ewing sarcoma cells infected with GGAAprom>Cas9 and a specific gRNA designed to inactivate EWSR1 : : FLI1, showed successful EWSR1 : : FLI1 inactivation and the subsequent blockade of cell proliferation. Notably, GGAAprom>Cas9 can be efficiently delivered to Ewing sarcoma cells via adenoviral vectors both in vitro and in vivo, highlighting the potential of this approach for Ewing sarcoma treatment. Our results demonstrate that the CRISPR/Cas9 machinery is safe and specific for Ewing sarcoma cells when driven under a GGAAprom, paving the way for the development of cancer gene therapies based on the selective expression of genes with therapeutic potential.