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Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells
Published on: September 25, 2019
CRISPR/Cas9-Mediated Gene Correction in Osteopetrosis Patient-Derived iPSCs
Dandan Li1,2, Minglin Ou1,3, Wei Zhang1,2
1Clinical Medical Research Center, The Second Clinical Medical College of Jinan University (Shenzhen People's Hospital), 518020 Shenzhen, Guangdong, China.
Background:
Osteopetrosis represents a rare genetic disease with a wide range of clinical and genetic heterogeneity, which results from osteoclast failure. Although up to 10 genes have been identified to be related with osteopetrosis, the pathogenesis of osteopetrosis remains foggy. Disease-specific induced pluripotent stem cells (iPSCs) and gene-corrected disease specific iPSCs provide a platform to generate attractive in vitro disease cell models and isogenic control cellular models respectively. The purpose of this study is to rescue the disease causative mutation in osteopetrosis specific induced pluripotent stem cells and provide isogenic control cellular models.
Methods:
Based on our previously established osteopetrosis-specific iPSCs (ADO2-iPSCs), we repaired the point mutation R286W of the CLCN7 gene in ADO2-iPSCs by the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) mediated homologous recombination.
Results:
The obtained gene corrected ADO2-iPSCs (GC-ADO2-iPSCs) were characterized in terms of hESC-like morphology, a normal karyotype, expression of pluripotency markers, homozygous repaired sequence of CLCN7 gene, and the ability to differentiate into cells of three germ layers.
Conclusions:
We successfully corrected the point mutation R286W of the CLCN7 gene in ADO2-iPSCs. This isogenic iPSC line is an ideal control cell model for deciphering the pathogenesis of osteopetrosis in future studies.
Insights
Researchers corrected a disease-causing mutation in osteopetrosis-specific induced pluripotent stem cells (iPSCs). This creates a valuable isogenic control cell model for studying osteopetrosis pathogenesis.
Area of Science:
- Genetics
- Stem Cell Biology
- Rare Diseases
Background:
- Osteopetrosis is a rare genetic disorder caused by osteoclast dysfunction, with complex and not fully understood pathogenesis.
- Despite identifying numerous genes, the precise mechanisms underlying osteopetrosis remain unclear.
- Induced pluripotent stem cells (iPSCs) offer a powerful tool for creating in vitro disease and isogenic control models.
Purpose of the Study:
- To correct the causative mutation in osteopetrosis-specific iPSCs.
- To generate isogenic control cellular models for osteopetrosis research.
- To facilitate future studies on the pathogenesis of osteopetrosis.
Main Methods:
- Utilized CRISPR/Cas9 gene editing technology to repair the R286W point mutation in the CLCN7 gene.
- Employed previously established osteopetrosis-specific iPSCs (ADO2-iPSCs) as the starting material.
- Performed homologous recombination for precise gene correction.
Main Results:
- Successfully generated gene-corrected ADO2-iPSCs (GC-ADO2-iPSCs).
- Confirmed normal karyotype, pluripotency marker expression, and differentiation potential into three germ layers.
- Verified homozygous repair of the CLCN7 gene mutation.
Conclusions:
- The CLCN7 gene mutation R286W in ADO2-iPSCs was effectively corrected.
- The resulting isogenic iPSC line serves as an ideal control model.
- This model will be instrumental in future research to elucidate osteopetrosis pathogenesis.
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