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Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells
Published on: September 25, 2019
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CRISPR Base Editing to Create Potential Charcot-Marie-Tooth Disease Models with High Editing Efficiency: Human
Camille Loret1, Amandine Pauset2,3, Pierre-Antoine Faye1,4
1University of Limoges, NeurIT UR 20218, GEIST Institute, F-87000 Limoges, France.
Biomedicines
|July 27, 2024
Summary
Researchers developed the first human induced pluripotent stem cell (hiPSC) models for Charcot-Marie-Tooth disease type 4C (CMT4C) using CRISPR editing. These models aid in studying the disease and testing therapies for this inherited peripheral neuropathy.
Area of Science:
- * Stem cell biology and regenerative medicine.
- * Genetic engineering and disease modeling.
- * Neuroscience and neurodegenerative disorders.
Background:
- * Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral neuropathy, with CMT4C being a prevalent demyelinating subtype.
- * Studying CMT is challenging due to the inaccessibility of affected peripheral nerve cells.
- * Human induced pluripotent stem cells (hiPSCs) offer a viable model system for investigating such disorders.
Purpose of the Study:
- * To generate the first hiPSC models for CMT4C, specifically targeting two distinct nonsense variants in the SH3TC2 gene.
- * To establish isogenic cellular models for studying the pathophysiology of AR-CMTde-SH3TC2.
- * To optimize CRISPR base-editing strategies for efficient genetic modification of hiPSCs.
Main Methods:
- * Utilized CRISPR base editing to introduce specific point mutations into hiPSCs.
- * Tested various sgRNA and base editor combinations in HEK-293T cells to optimize editing strategies.
- * Applied the optimized CRISPR strategy to hiPSCs from healthy individuals to create CMT4C models.
Main Results:
- * Successfully generated hiPSC models for two SH3TC2 nonsense variants (c.211C>T, p.Gln71* and c.2860C>T, p.Arg954*) associated with CMT4C.
- * Achieved high editing efficiency (up to 93%) in hiPSCs using the optimized CRISPR base-editing approach.
- * Demonstrated the utility of HEK-293T cells for pre-screening CRISPR strategies before application to hiPSCs.
Conclusions:
- * Efficient generation of isogenic hiPSC models for CMT4C is feasible using CRISPR base editing.
- * Optimizing CRISPR strategies in easily transfectable cell lines like HEK-293T is recommended prior to hiPSC editing.
- * These novel hiPSC models provide valuable tools for advancing CMT4C research and therapeutic development.
