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

Fragile X syndrome (FXS) is a genetic disease with intellectual disabilities. FXS is often caused by the CGG-repeat expansion mutation in the FMR1gene with suppressed FMR1transcription and decreased protein levels in the brain of the patients. The RNA-guided CRISPR/Cas9system is a promising targeted ge-nomic editing tool in gene therapy of FXS. In order to evaluate its feasibility, the present study used CRISPR/Cas9system to target the FMR15’-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 t-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 (P<0.05). In the knockout cells, apoptosis was accelerated (P<0.05) with a significantlydown-regulated (P<0.05) expression of FMRP as compared with the control group.

This study provides further understanding about the FMRP function and molecular mechanism of FMR1gene in nerve cells, and suggests the feasibility of gene therapyin FXS by CRISPR/Cas9gene editing system.