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

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. Access through your institution

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OTHERS CORRECTION OF EXON 2, EXON 2–9 AND EXONS 8–9 DUPLICATIONS IN DMD PATIENT MYOGENIC CELLS BY A SINGLE CRISPR/CAS9 SYSTEM Article Open access 11 September 2024 TRANSIENTLY EXPRESSED

CRISPR/CAS9 INDUCES WILD-TYPE DYSTROPHIN IN VITRO IN DMD PATIENT MYOBLASTS CARRYING DUPLICATIONS Article Open access 08 March 2022 GENOME EDITING FOR DUCHENNE MUSCULAR DYSTROPHY: A GLIMPSE

OF THE FUTURE? Article Open access 02 February 2021 DATA AVAILABILITY The data generated or analyzed during this study are presented in the main paper and the supplementary file. Additional

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2020;11:1334. Article  CAS  Google Scholar  Download references 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. AUTHOR INFORMATION AUTHORS AND

AFFILIATIONS * Department of Neurology, The First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, China Dan-Ni Wang * Department of Neurology and

Institute of Neurology of First Affiliated Hospital, Institute of Neuroscience, and Fujian Key Laboratory of Molecular Neurology, Fujian Medical University, Fuzhou, China Zhi-Qiang Wang, 

Ming Jin, Min-Ting Lin & Ning Wang Authors * Dan-Ni Wang View author publications You can also search for this author inPubMed Google Scholar * Zhi-Qiang Wang View author publications

You can also search for this author inPubMed Google Scholar * Ming Jin View author publications You can also search for this author inPubMed Google Scholar * Min-Ting Lin View author

publications You can also search for this author inPubMed Google Scholar * Ning Wang View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS 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. CORRESPONDING AUTHOR

Correspondence to Ning Wang. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with

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ARTICLE CITE THIS ARTICLE 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_ 29, 730–737 (2022). https://doi.org/10.1038/s41434-022-00336-3 Download citation * Received: 08 October 2021 * Revised: 29 March 2022 * Accepted: 06 April 2022 * Published: 09 May 2022

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