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Updated: May 26, 2025
Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells
Published on: September 25, 2019
Generation of a human iPSC line with Notch3 R133C mutation by CRISPR/Cas9: A tool for investigating CADASIL and
Sema Aygar1, Laurence Daheron1
1Harvard University Harvard Stem Cell Institute IPS Core Facility Cambridge USA.
Abstract:
Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a rare neuro vascular disease that is caused by mutations in Notch3. Here, we developed an iPSC line harboring the R133C mutation in Notch3, which is among the most common mutations leading to CADASIL. This mutation alters the disulfide bonding pattern leading to Notch3 protein aggregation, granular osmiophilic material (GOM) formation and vascular changes. The iPSC line was generated using CRISPR/Cas9 and edits were confirmed by PCR and Sanger sequencing. This resource is a valuable tool for studying molecular mechanisms of CADASIL and enabling the development and screening of targeted therapies for Notch3-related pathologies.
Insights
Researchers created a new induced pluripotent stem cell (iPSC) line with a common Notch3 mutation. This iPSC line models cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) for disease research.
Area of Science:
- Neuroscience
- Genetics
- Stem Cell Biology
Background:
- Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a rare, inherited cerebrovascular disorder.
- CADASIL is primarily caused by mutations in the Notch3 gene, affecting vascular smooth muscle cells.
Purpose of the Study:
- To develop a novel induced pluripotent stem cell (iPSC) line harboring a specific Notch3 mutation (R133C) associated with CADASIL.
- To provide a valuable research tool for investigating the molecular pathogenesis of CADASIL.
Main Methods:
- Utilized CRISPR/Cas9 gene editing technology to introduce the R133C mutation into Notch3 within iPSCs.
- Confirmed the successful gene edit through Polymerase Chain Reaction (PCR) and Sanger sequencing.
Main Results:
- Successfully generated and validated an iPSC line carrying the Notch3 R133C mutation.
- The mutation is known to disrupt Notch3 protein structure, leading to aggregation and granular osmiophilic material (GOM) formation.
Conclusions:
- The developed iPSC line serves as a crucial in vitro model for studying CADASIL.
- This resource facilitates the exploration of Notch3-related pathologies and the screening of potential therapeutic interventions.
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