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Updated: Aug 20, 2025
CRISPR/Cas9 Technology in Restoring Dystrophin Expression in iPSC-Derived Muscle Progenitors
Published on: September 14, 2019
CRISPR-Cas9 Correction of Duchenne Muscular Dystrophy in Mice by a Self-Complementary AAV Delivery System
Yu Zhang1,2,3, Rhonda Bassel-Duby1,2,3, Eric N Olson4,5,6
1Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA.
Abstract:
Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disorder, caused by mutations in the DMD gene coding dystrophin. Applying clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (CRISPR-Cas) for therapeutic gene editing represents a promising technology to correct this devastating disease through elimination of underlying genetic mutations. Adeno-associated virus (AAV) has been widely used for gene therapy due to its low immunogenicity and high tissue tropism. In particular, CRISPR-Cas9 gene editing components packaged by self-complementary AAV (scAAV) demonstrate robust viral transduction and efficient gene editing, enabling restoration of dystrophin expression throughout skeletal and cardiac muscle in animal models of DMD. Here, we describe protocols for cloning CRISPR single guide RNAs (sgRNAs) into a scAAV plasmid and procedures for systemic delivery of AAVs into a DMD mouse model. We also provide methodologies for quantification of dystrophin restoration after systemic CRISPR-Cas9-mediated correction of DMD.
Insights
Gene editing using CRISPR-Cas9 delivered by adeno-associated virus (AAV) shows promise for Duchenne muscular dystrophy (DMD). This method effectively restores dystrophin expression in preclinical models, offering a potential therapeutic strategy for DMD patients.
Area of Science:
- Biotechnology
- Gene Therapy
- Molecular Biology
Background:
- Duchenne muscular dystrophy (DMD) is a severe, fatal genetic neuromuscular disorder caused by mutations in the DMD gene.
- Current treatments for DMD are limited, highlighting the need for innovative therapeutic approaches.
Purpose of the Study:
- To develop and validate protocols for CRISPR-Cas9 gene editing delivery via self-complementary adeno-associated virus (scAAV) for DMD.
- To assess the efficacy of systemic AAV-CRISPR-Cas9 delivery in restoring dystrophin expression in a mouse model of DMD.
Main Methods:
- Cloning of CRISPR single guide RNAs (sgRNAs) into a scAAV plasmid.
- Systemic administration of AAV vectors into a mouse model of Duchenne muscular dystrophy.
- Quantification of dystrophin restoration in skeletal and cardiac muscle tissues.
Main Results:
- Successful packaging of CRISPR-Cas9 gene editing components into scAAV vectors.
- Demonstrated robust viral transduction and efficient gene editing in the DMD mouse model.
- Significant restoration of dystrophin expression in skeletal and cardiac muscles following systemic AAV delivery.
Conclusions:
- Systemic delivery of CRISPR-Cas9 via scAAV is a viable strategy for correcting the genetic defect in Duchenne muscular dystrophy.
- This gene editing approach holds potential for restoring dystrophin production and treating DMD in preclinical settings.
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