i3S-Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135 Porto, Portugal.
Cancer Signaling and Metabolism Group, IPATIMUP-Institute of Molecular Pathology and Immunology of the University of Porto, Rua Alfredo Allen 208, 4169-007 Porto, Portugal.
Int J Mol Sci. 2023 Jul 10;24(14):11285. doi: 10.3390/ijms241411285.
remains one of the most altered genes in cancer, although its relevance in cancer biology is still unclear. Recent advances in gene editing techniques, particularly CRISPR/Cas9 systems, offer new opportunities to evaluate the function of large genes, such as . Using a dual sgRNA CRISPR/Cas9 gene editing approach, this study aimed to assess the impact of disrupting in glioblastoma cell biology. Four sgRNAs were designed for the dual targeting of two exons (1 and 85). The U87 glioblastoma (GB) cell line was transfected with CRISPR/Cas9 PX459 vectors. To assess -gene-induced alterations and expression, PCR, Sanger DNA sequencing, and qRT-PCR were carried out. Three clones (clones B9, E6, and H7) were further evaluated. All clones presented altered cellular morphology, increased cellular and nuclear size, and changes in ploidy. Two clones (E6 and H7) showed a significant decrease in cell growth, both in vitro and in the in vivo CAM assay. Proteomic analysis of the clones' secretome identified differentially expressed proteins that had not been previously associated with alterations. This study demonstrates that the dual sgRNA CRISPR/Cas9 strategy can effectively edit in GB cells, providing new insights into the impact of deletions in GBM biology.
仍然是癌症中改变最多的基因之一，尽管其在癌症生物学中的相关性仍不清楚。基因编辑技术的最新进展，特别是 CRISPR/Cas9 系统，为评估大型基因的功能提供了新的机会，如 。本研究采用双 sgRNA CRISPR/Cas9 基因编辑方法，旨在评估破坏 对神经胶质瘤细胞生物学的影响。设计了四个 sgRNA 用于靶向两个 外显子（1 和 85）的双重靶向。U87 神经胶质瘤（GB）细胞系用 CRISPR/Cas9 PX459 载体转染。为了评估 -基因诱导的改变和表达，进行了 PCR、Sanger DNA 测序和 qRT-PCR。进一步评估了三个克隆（克隆 B9、E6 和 H7）。所有克隆均呈现出改变的细胞形态、增大的细胞和核大小以及倍性变化。两个克隆（E6 和 H7）显示出体外和体内 CAM 测定中细胞生长的显著下降。克隆分泌组的蛋白质组学分析鉴定出以前与 改变无关的差异表达蛋白。本研究表明，双 sgRNA CRISPR/Cas9 策略可有效编辑 GB 细胞中的 ，为 缺失对 GBM 生物学的影响提供了新的见解。
