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CRISPR/Cas9-based genome editing for the modification of multiple duplications that cause Duchenne muscular dystrophy

Abstract

With the development of basic research, some genetic-based methods have been found to treat Duchenne muscular dystrophy (DMD) with large deletion mutations and nonsense mutations. Appropriate therapeutic approaches for repairing multiple duplications are limited. We used the CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 system with patient-derived primary myoblasts to correct multiple duplications of the dystrophin gene. Muscle tissues from a patient carrying duplications of dystrophin were obtained, and tissue-derived primary cells were cultured. Myoblasts were purified with an immunomagnetic sorting system using CD56 microbeads. After transduction by lentivirus with a designed single guide RNA (sgRNA) targeting a duplicated region, myoblasts were allowed to differentiate for 7 days. Copy number variations in the exons of the patient’s myotubes were quantified by real-time PCR before and after genetic editing. Western blot analysis was performed to detect the full-length dystrophin protein before and after genetic editing. The ten sequences predicted to be the most likely off-targets were determined by Sanger sequencing. The patient carried duplications of exon 18–25, dystrophin protein expression was completely abrogated. Real-time PCR showed that the copy number of exon 25 in the patient’s myotubes was 2.015 ± 0.079 compared with that of the healthy controls. After editing, the copy number of exon 25 in the patient’s modified myotubes was 1.308 ± 0.083 compared with that of the healthy controls (P < 0.001). Western blot analysis revealed no expression of the dystrophin protein in the patient’s myotubes before editing. After editing, the patient’s myotubes expressed the full-length dystrophin protein at a level that was ~6.12% of that in the healthy control samples. Off-target analysis revealed no abnormal editing at the ten sites predicted to be the most likely off-target sites. The excision of multiple duplications by the CRISPR/Cas9 system restored the expression of full-length dystrophin. This study provides proof of evidence for future genome-editing therapy in patients with DMD caused by multiple duplication mutations.

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Fig. 1: Detection of dystrophin gene mutation in the patient with DMD.
Fig. 2: Results of magnetic cell sorting.
Fig. 3: Digestion results of T7 endonuclease.
Fig. 4: Real-time quantitative PCR results.
Fig. 5: Western blot analysis of dystrophin protein before and after dystrophin gene editing.

Data availability

The data generated or analyzed during this study are presented in the main paper and the supplementary file. Additional data are available from the corresponding author on reasonable request.

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Funding

This work was supported by the grant 81801248 from the National Natural Science Foundation of China, grant 81870902 from the National Natural Science Foundation of China, and the Joint Funds 2018Y9082 for the Innovation of Science and Technology of Fujian province.

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NW conceived and initiated the research; NW and Z-QW directed the research; NW and D-NW designed the experiments; D-NW and M-TL generated and characterized reagents and tools; D-NW performed all the experiments; D-NW and Z-QW analyzed the data together with all authors; D-NW wrote the paper with the aid of NW. All the authors read and approved the paper.

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Correspondence to Ning Wang.

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Wang, DN., Wang, ZQ., Jin, M. et al. CRISPR/Cas9-based genome editing for the modification of multiple duplications that cause Duchenne muscular dystrophy. Gene Ther (2022). https://doi.org/10.1038/s41434-022-00336-3

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  • DOI: https://doi.org/10.1038/s41434-022-00336-3

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