Accelerated Apoptosis and Down-Regulated FMRP in Human Neuroblastoma Cells with CRISPR/Cas9 Genome Editing

BACKGROUND: Fragile X syndrome (FXS) is a genetic disease with intellectual disabilities. FXS is often caused by the CGG-repeat expansion mutation in the FMR1 gene with suppressed FMR1 transcription and decreased protein levels in the brain of the patients. The RNA-guided CRISPR/Cas9 system is a promising targeted genomic editing tool in gene therapy of FXS. In order to evaluate its feasibility, the present study used CRISPR/Cas9 system to target the FMR1 5′-UTR sites in cultured human neuroblastoma cells. METHODS: 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 t-test using GraphPad software. This research was conducted from 2020 to 2022 in the Second Affiliated Hospital of Soochow University, Suzhou, China. RESULTS: Cell cycle analysis showed significant differences in G1, S and G2/M phases between the two groups (P<0.05). In the knockout cells, apoptosis was accelerated (P<0.05) with a significantly down-regulated (P<0.05) expression of FMRP as compared with the control group. CONCLUSION: This study provides further understanding about the FMRP function and molecular mechanism of FMR1 gene in nerve cells, and suggests the feasibility of gene therapy in FXS by CRISPR/Cas9 gene editing system.