Accelerated Apoptosis and Down-Regulated FMRP in Human Neuroblastoma Cells with CRISPR/ 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 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.

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 -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 (<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.

CONCLUSION

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.