Related Experiment Video
Updated: Jul 19, 2025
Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells
Published on: January 5, 2018
Accelerated Apoptosis and Down-Regulated FMRP in Human Neuroblastoma Cells with CRISPR/Cas9 Genome Editing
Rong Zhang1,2, Huifen Xu3, Jin Lu1
1Department of Gynecology and Obstetrics, the Second Affiliated Hospital of Soochow University, Suzhou, 215004, China.
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.
Insights
CRISPR/Cas9 gene editing successfully targeted the FMR1 gene in human cells, offering a potential therapy for Fragile X syndrome (FXS). This approach reduced FMRP protein and altered cell cycle, supporting its feasibility for FXS treatment.
Area of Science:
- Genetics
- Molecular Biology
- Neuroscience
Background:
- Fragile X syndrome (FXS) is a genetic disorder causing intellectual disabilities, often due to CGG-repeat expansion in the FMR1 gene.
- This mutation leads to reduced FMR1 gene transcription and FMRP protein levels in the brain.
- The CRISPR/Cas9 system presents a potential gene therapy strategy for FXS.
Purpose of the Study:
- To evaluate the feasibility of using the CRISPR/Cas9 system for gene therapy in FXS.
- To target the FMR1 gene's 5'-UTR in cultured human neuroblastoma cells.
Main Methods:
- Plasmid construction using PCR and DNA cloning.
- CRISPR/Cas9 function assessment via Western blot and flow cytometry.
- Statistical analysis using two-tailed unpaired Student's t-test.
Main Results:
- CRISPR/Cas9 targeting of FMR1 resulted in significant differences in cell cycle phases (G1, S, G2/M).
- Apoptosis was accelerated in knockout cells.
- FMRP expression was significantly downregulated in knockout cells compared to controls.
Conclusions:
- The study enhances understanding of FMRP function and the molecular mechanisms of the FMR1 gene in nerve cells.
- Findings suggest the feasibility of CRISPR/Cas9 gene editing as a therapeutic approach for Fragile X syndrome.
Related Concept Videos
01:59CRISPR
01:28CRISPR/Cas9 Genome Editing