文章：

由Ewing特异性GGAA启动子驱动的CRISPR/Cas9技术靶向Ewing肉瘤中的FLI1基因

FLI1 gene in Ewing sarcoma via CRISPR/Cas9 driven by an Ewing-specific GGAA promoter 

原文发布日期：2025-03-15 

英文摘要：

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. 

摘要翻译：

我们近期通过CRISPR/Cas9技术实现EWSR1::FLI1基因失活的研究表明，该方法能成功抑制尤文肉瘤细胞模型的增殖。然而CRISPR/Cas9介导的基因编辑可能存在脱靶效应，因此精确调控靶细胞中Cas9的表达对于开发尤文肉瘤细胞EWSR1::FLI1失活的基因编辑策略至关重要。本研究发现，当将Cas9置于由GGAA重复序列和TATA盒保守序列（GGAAprom）组成的启动子下游时，能在尤文肉瘤细胞中实现特异性表达。在此条件下，Cas9仅在选择性表达EWSR1::FLI1癌蛋白的尤文肉瘤细胞中表达，而在表达野生型FLI1的细胞中不表达。随之，感染GGAAprom>Cas9和特异性gRNA（用于失活EWSR1::FLI1）的尤文肉瘤细胞成功实现了EWSR1::FLI1失活及后续的增殖阻滞。值得注意的是，腺病毒载体可在体外和体内高效递送GGAAprom>Cas9至尤文肉瘤细胞，这凸显了该疗法在尤文肉瘤治疗中的潜力。我们的研究结果表明，由GGAAprom驱动的CRISPR/Cas9系统对尤文肉瘤细胞具有安全性和特异性，为开发基于治疗性基因选择性表达的癌症基因疗法奠定了基础。

原文链接：

FLI1 gene in Ewing sarcoma via CRISPR/Cas9 driven by an Ewing-specific GGAA promoter