Zhang Rong, Xu Huifen, Lu Jin, Chen Ying, Zhang Yahui, Xiao Li
Department of Gynecology and Obstetrics, the Second Affiliated Hospital of Soochow University, Suzhou, 215004, China.
State Key Laboratory of Radiation Medicine and Protection, Soochow University, Suzhou, 215123, China.
Iran J Public Health. 2023 Apr;52(4):703-712. doi: 10.18502/ijph.v52i4.12438.
Fragile X syndrome (FXS) is a genetic disease with intellectual disabilities. FXS is often caused by the CGG-repeat expansion mutation in the gene with suppressed transcription and decreased protein levels in the brain of the patients. The RNA-guided CRISPR/ system is a promising targeted genomic editing tool in gene therapy of FXS. In order to evaluate its feasibility, the present study used CRISPR/ system to target the 5'-UTR sites in cultured human neuroblastoma cells.
PCR and DNA clone were used to construct plasmids. CRISPR function was tested by Western blot and flow cytometry. Data were analyzed by a two-tailed unpaired Student's -test using GraphPad software. This research was conducted from 2020 to 2022 in the Second Affiliated Hospital of Soochow University, Suzhou, China.
Cell cycle analysis showed significant differences in G1, S and G2/M phases between the two groups (<0.05). In the knockout cells, apoptosis was accelerated (<0.05) with a significantly down-regulated (<0.05) expression of FMRP as compared with the control group.
This study provides further understanding about the FMRP function and molecular mechanism of gene in nerve cells, and suggests the feasibility of gene therapy in FXS by CRISPR/ gene editing system.
脆性X综合征（FXS）是一种伴有智力障碍的遗传性疾病。FXS通常由基因中的CGG重复扩增突变引起，患者大脑中该基因转录受抑制且蛋白质水平降低。RNA引导的CRISPR/Cas系统是FXS基因治疗中一种有前景的靶向基因组编辑工具。为评估其可行性，本研究使用CRISPR/Cas系统靶向培养的人神经母细胞瘤细胞中的5'-UTR位点。
采用PCR和DNA克隆构建质粒。通过蛋白质免疫印迹法和流式细胞术检测CRISPR功能。使用GraphPad软件通过双尾非配对学生t检验分析数据。本研究于2020年至2022年在中国苏州的苏州大学附属第二医院进行。
细胞周期分析显示两组在G1、S和G2/M期存在显著差异（P<0.05）。与对照组相比，在基因敲除细胞中，凋亡加速（P<0.05），脆性X智力低下蛋白（FMRP）表达显著下调（P<0.05）。
本研究进一步了解了FMRP在神经细胞中的功能以及该基因的分子机制，并表明CRISPR/Cas基因编辑系统在FXS基因治疗中的可行性。
