CRISPR technologies for the treatment of Duchenne muscular dystrophy
Authors: Eunyoung Choi, Taeyoung Koo
Journal: Molecular Therapy (2021)

The emerging clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome editing technologies have progressed remarkably in recent years, opening up the potential of precise genome editing as a therapeutic approach to treat various diseases. The CRISPR-CRISPR-associated (Cas) system is an attractive platform for the treatment of Duchenne muscular dystrophy (DMD), which is a neuromuscular disease caused by mutations in the DMD gene. CRISPR-Cas can be used to permanently repair the mutated DMD gene, leading to the expression of the encoded protein, dystrophin, in systems ranging from cells derived from DMD patients to animal models of DMD. However, the development of more efficient therapeutic approaches and delivery methods remains a great challenge for DMD. Here, we review various therapeutic strategies that use CRISPR-Cas to correct or bypass DMD mutations and discuss their therapeutic potential, as well as obstacles that lie ahead. The emerging clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome editing technologies have progressed remarkably in recent years, opening up the potential of precise genome editing as a therapeutic approach to treat various diseases. The CRISPR-CRISPR-associated (Cas) system is an attractive platform for the treatment of Duchenne muscular dystrophy (DMD), which is a neuromuscular disease caused by mutations in the DMD gene. CRISPR-Cas can be used to permanently repair the mutated DMD gene, leading to the expression of the encoded protein, dystrophin, in systems ranging from cells derived from DMD patients to animal models of DMD. However, the development of more efficient therapeutic approaches and delivery methods remains a great challenge for DMD. Here, we review various therapeutic strategies that use CRISPR-Cas to correct or bypass DMD mutations and discuss their therapeutic potential, as well as obstacles that lie ahead. IntroductionDuchenne muscular dystrophy (DMD) is a severe, X-linked recessive disease with an average incidence of ∼1 in 5,000 live male births.1Sussman M. Duchenne muscular dystrophy.J. Am. Acad. Orthop. Surg. 2002; 10: 138-151Crossref PubMed Scopus (105) Google Scholar Most DMD patients exhibit progressive muscle degeneration associated with severe muscle weakness, loss of ambulation, cardiac or respiratory complications, and eventually death, in their 20s.2Cai A. Kong X. Development of CRISPR-Mediated Systems in the Study of Duchenne Muscular Dystrophy.Hum. Gene Ther. Methods. 2019; 30: 71-80Crossref PubMed Scopus (9) Google ScholarThe DMD gene consists of 79 exons that encode dystrophin, which is a cytoskeletal protein that plays an important role in a complex that connects the cytoskeleton of muscle fibers with the extracellular matrix and is present throughout the cell membrane.3Lim K.R.Q. Yoon C. Yokota T. Applications of CRISPR/Cas9 for the treatment of duchenne muscular dystrophy.J. Pers. Med. 2018; 8: 38Crossref Scopus (33) Google Scholar, 4Roberts R.G. Coffey A.J. Bobrow M. Bentley D.R. Exon structure of the human dystrophin gene.Genomics. 1993; 16: 536-538Crossref PubMed Scopus (143) Google Scholar, 5Hoffman E.P. Brown Jr., R.H. Kunkel L.M. Dystrophin: the protein product of the Duchenne muscular dystrophy locus.Cell. 1987; 51: 919-928Abstract Full Text PDF PubMed Scopus (3402) Google Scholar Different types of mutations in DMD exons and introns cause various forms of dystrophinopathies.3Lim K.R.Q. Yoon C. Yokota T. Applications of CRISPR/Cas9 for the treatment of duchenne muscular dystrophy.J. Pers. Med. 2018; 8: 38Crossref Scopus (33) Google Scholar Approximately 60% of DMD patients harbor a large deletion in the DMD gene, often affecting exons 45–55, a region that represents a mutational hotspot.6Bladen C.L. Salgado D. Monges S. Foncuberta M.E. Kekou K. Kosma K. Dawkins H. Lamont L. Roy A.J. Chamova T. et al.The TREAT-NMD DMD Global Database: analysis of more than 7,000 Duchenne muscular dystrophy mutations.Hum. Mutat. 2015; 36: 395-402Crossref PubMed Scopus (306) Google Scholar Deletion of a DMD exon can result in a shift in the reading frame and the formation of a premature stop codon, causing either expression of a truncated version of dystrophin that does not function properly or a complete lack of dystrophin expression.In Becker muscular dystrophy (BMD), a relatively benign type of MD compared to DMD, a semi-functional dystrophin protein is expressed, compensating for the muscle loss.2Cai A. Kong X. Development of CRISPR-Mediated Systems in the Study of Duchenne Muscular Dystrophy.Hum. Gene Ther. Methods. 2019; 30: 71-80Crossref PubMed Scopus (9) Google Scholar Mild BMD symptoms include a relatively slow disease progression, which have little effect on lifespan.7Monaco A.P. Bertelson C.J. Liechti-Gallati S. Moser H. Kunkel L.M. An explanation for the phenotypic differences between patients bearing partial deletions of the DMD locus.Genomics. 1988; 2: 90-95Crossref PubMed Scopus (869) Google Scholar Thus, alleviating symptoms in DMD patients by expressing a semi-functional protein to mimic a BMD-like disease phenotype could be an efficient strategy for treating DMD. It is notable that a 4% increase in normal dystrophin expression was sufficient to improve muscle function.8van Putten M. Hulsker M. Young C. Nadarajah V.D. Heemskerk H. van der Weerd L. et al.Low dystrophin levels increase survival and improve muscle pathology and function in dystrophin/utrophin double-knockout mice.FASEB J. 2013; 27: 2484-2495Crossref PubMed Scopus (66) Google Scholar, 9Li D. Yue Y. Duan D. Marginal level dystrophin expression improves clinical outcome in a strain of dystrophin/utrophin double knockout mice.PLoS ONE. 2010; 5: e15286Crossref PubMed Scopus (38) Google Scholar, 10van Putten M. van der Pijl E.M. Hulsker M. Verhaart I.E.C. Nadarajah V.D. van der Weerd L. et al.Low dystrophin levels in heart can delay heart failure in mdx mice.J. Mol. Cell. Cardiol. 2014; 69: 17-23Abstract Full Text Full Text PDF PubMed Google ScholarVarious pharmacologic therapeutic approaches have focused on converting the DMD phenotype to a BMD-like phenotype by restoring the disrupted DMD reading frame. In 2016, eteplirsen (Exondys 51), an antisense oligonucleotide drug with phosphorodiamidate morpholino oligomer chemistry, became the first medication with such a mechanism to be approved by the US Food and Drug Administration (FDA) for the treatment of DMD. It induces exon 51 skipping in the DMD gene, restoring the expression of semi-functional dystrophin and resulting in BMD-like mild symptoms in eteplirsen-treated DMD patients.11Heo Y.A. Golodirsen: First Approval.Drugs. 2020; 80: 329-333Crossref PubMed Scopus (65) Google Scholar All of the patients treated with eteplirsen showed an increase in the frequency of dystrophin-positive fibers, by an average of 15.5-fold over untreated controls. In addition, the therapeutic efficacy of golodirsen (Vyondys 53) and viltolarsen (Viltepso), other phosphorodiamidate morpholino oligomer drugs, are under evaluation in clinical trials for treating DMD patients.12Clemens P.R. Rao V.K. Connolly A.M. Harper A.D. Mah J.K. Smith E.C. McDonald C.M. Zaidman C.M. Morgenroth L.P. Osaki H. et al.CINRG DNHS InvestigatorsSafety, tolerability, and efficacy of viltolarsen in boys with duchenne muscular dystrophy amenable to exon 53 skipping: A phase 2 randomized clinical trial.JAMA Neurol. 2020; 77: 982-991Crossref PubMed Scopus (18) Google Scholar,13Komaki H. Nagata T. Saito T. Masuda S. Takeshita E. Sasaki M. Tachimori H. Nakamura H. Aoki Y. Takeda S. Systemic administration of the antisense oligonucleotide NS-065/NCNP-01 for skipping of exon 53 in patients with Duchenne muscular dystrophy.Sci. Transl. Med. 2018; 10: eaan0713Crossref PubMed Scopus (36) Google Scholar Treatment with golodirsen in a Phase I trial resulted in exon 53 skipping and a ∼16-fold increase in dystrophin protein expression over baseline, with 1.02% of normal dystrophin protein expression at week 48. In another study, a 4-week randomized Phase II clinical trial, treatment with viltolarsen caused exon 53 skipping and transcript levels that were 42.4% of normal levels, which in turn led to significant dystrophin production, at 2.8% of normal levels.12Clemens P.R. Rao V.K. Connolly A.M. Harper A.D. Mah J.K. Smith E.C. McDonald C.M. Zaidman C.M. Morgenroth L.P. Osaki H. et al.CINRG DNHS InvestigatorsSafety, tolerability, and efficacy of viltolarsen in boys with duchenne muscular dystrophy amenable to exon 53 skipping: A phase 2 randomized clinical trial.JAMA Neurol. 2020; 77: 982-991Crossref PubMed Scopus (18) Google Scholar, 13Komaki H. Nagata T. Saito T. Masuda S. Takeshita E. Sasaki M. Tachimori H. Nakamura H. Aoki Y. Takeda S. Systemic administration of the antisense oligonucleotide NS-065/NCNP-01 for skipping of exon 53 in patients with Duchenne muscular dystrophy.Sci. Transl. Med. 2018; 10: eaan0713Crossref PubMed Scopus (36) Google Scholar, 14Frank D.E. Schnell F.J. Akana C. El-Husayni S.H. Desjardins C.A. Morgan J. Charleston J.S. Sardone V. Domingos J. Dickson G. et al.SKIP-NMD Study GroupIncreased dystrophin production with golodirsen in patients with Duchenne muscular dystrophy.Neurology. 2020; 94: e2270-e2282Crossref PubMed Scopus (64) Google Scholar These approaches have reduced disease symptoms, but none have yet eliminated the disease-causing mutation to allow long-term dystrophin expression.Recently, therapeutic applications of genome editing have been explored for treating various genetic diseases. The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) system is a powerful technology for genome editing, especially for correcting disease-causing mutations. Here, we review recent progress in the area of CRISPR-mediated genome editing to treat DMD with various strategies to eliminate pathogenic mutations in the DMD gene. These approaches could also be extensively applied for treating other neuromuscular disorders and other types of genetic diseases.Genome editing tools: The CRISPR-Cas systemCRISPR-Cas was identified as an adaptive immune system in bacteria and archaea that functions to prevent the invasion of foreign genetic materials.15Jinek M. Chylinski K. Fonfara I. Hauer M. Doudna J.A. Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science. 2012; 337: 816-821Crossref PubMed Scopus (7412) Google Scholar Upon viral DNA entry into a bacterium, the cell integrates segments of viral DNA into the CRISPR locus of a bacterial genome. When the same type of virus next invades, RNAs are transcribed from the CRISPR array and cooperate with Cas9 endonuclease to cleave the complementary viral DNA sequence. In the CRISPR system, two RNAs (CRISPR RNA [crRNA] and trans-activating CRISPR RNA [tracrRNA]) are transcribed for target searching. These two components can be linked together to generate a programmable single guide RNA (sgRNA), the form that is now widely used for efficient genome editing. Cas9 derived from the type II CRISPR system of Streptococcus pyogenes (SpCas9) is the most studied and generally used form of the endonuclease; it recognizes a 5′-NGG-3′ or 5′-NAG-3′ protospacer adjacent motif (PAM) and cleaves target DNA 3 bp upstream of the PAM, generating double-strand breaks (DSBs)15Jinek M. Chylinski K. Fonfara I. Hauer M. Doudna J.A. Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science. 2012; 337: 816-821Crossref PubMed Scopus (7412) Google Scholar, 16Hsu P.D. Scott D.A. Weinstein J.A. Ran F.A. Konermann S. Agarwala V. Li Y. Fine E.J. Wu X. Shalem O. et al.DNA targeting specificity of RNA-guided Cas9 nucleases.Nat. Biotechnol. 2013; 31: 827-832Crossref PubMed Scopus (2581) Google Scholar, 17Zhang Y. Ge X. Yang F. Zhang L. Zheng J. Tan X. Jin Z.B. Qu J. Gu F. Comparison of non-canonical PAMs for CRISPR/Cas9-mediated DNA cleavage in human cells.Sci. Rep. 2014; 4: 5405Crossref PubMed Scopus (127) Google Scholar (Figure 1A). Over time, Cas proteins from various other species have been discovered. Among them, Cas12a endonuclease, also called CRISPR from Prevotella and Francisella 1 (Cpf1), is derived from a type V (class II) CRISPR system.18Zetsche B. Gootenberg J.S. Abudayyeh O.O. Slaymaker I.M. Makarova K.S. Essletzbichler P. Volz S.E. Joung J. van der Oost J. Regev A. et al.Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system.Cell. 2015; 163: 759-771Abstract Full Text Full Text PDF PubMed Google Scholar It has been reported that Cpf1 endonuclease from Acidaminococcus sp. BV3L6 and Lachnospiraceae bacterium ND2006 recognize T-rich PAM motifs (5′-TTTV-3′) and cause staggered end cleavage with equal or greater efficiency than Cas9 orthologs.19Tóth E. Weinhardt N. Bencsura P. Huszár K. Kulcsár P.I. Tálas A. Fodor E. Welker E. Cpf1 nucleases demonstrate robust activity to induce DNA modification by exploiting homology directed repair pathways in mammalian cells.Biol. Direct. 2016; 11: 46Crossref PubMed Scopus (39) Google Scholar Of special interest, Cpf1 can be used for multiplex genome editing, in which multiple sites are simultaneously edited. Other Cas9 orthologs derived from Staphylococcus aureus (SaCas9)20Ran F.A. Cong L. Yan W.X. Scott D.A. Gootenberg J.S. Kriz A.J. Zetsche B. Shalem O. Wu X. Makarova K.S. et al.In vivo genome editing using Staphylococcus aureus Cas9.Nature. 2015; 520: 186-191Crossref PubMed Scopus (1450) Google Scholar or Campylobacter jejuni (CjCas9),21Kim E. Koo T. Park S.W. Kim D. Kim K. Cho H.Y. Song D.W. Lee K.J. Jung M.H. Kim S. et al.In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni.Nat. Commun. 2017; 8: 14500Crossref PubMed Scopus (280) Google Scholar which are smaller than SpCas9, make it possible to efficiently package the genes encoding these nucleases into small viral vector systems together with its sgRNA. The 2 Cas9 nucleases respectively recognize 5′-NNGRRT-3′ and 5′-NNNVRYAC-3′ PAM sequence before target DNA cleavage.More recently, several base editing systems have been developed that allow single base conversion or base editing in cells and organisms in a guide RNA-dependent manner. For targeted base mutagenesis, fusion of a deaminase enzyme, activation-induced cytidine deaminase (AID) or rat APOBEC1, with a catalytically deficient D10A/H840A Cas9 (called dead Cas9 or dCas9) or Cas9 nickase (nCas9) and uracil DNA glycosylase inhibitor (UGI, to prevent base excision repair) enabled direct conversion of a targeted cytidine (C):guanine (G) base pairs to thymine (T):adenine (A) base pairs (Figure 1B).22Ma Y. Zhang J. Yin W. Zhang Z. Song Y. Chang X. 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Scarfò I. et al.Engineered CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing.Nat. Biotechnol. 2019; 37: 276-282Crossref PubMed Scopus (150) Google Scholar Although the CRISPR-Cas and base editing systems can precisely install or correct mutations, they have limitations; in particular, Cas9 activity can lead to transversions and random insertions or deletions (indels) at the target site. Moreover, base editors can generate undesired bystander mutations within the base editing window. Most recently, a new genome editing technology, prime editors, was developed with the potential to overcome the limitations of the current genome editing system.36Anzalone A.V. Randolph P.B. Davis J.R. Sousa A.A. Koblan L.W. Levy J.M. Chen P.J. Wilson C. Newby G.A. Raguram A. Liu D.R. Search-and-replace genome editing without double-strand breaks or donor DNA.Nature. 2019; 576: 149-157Crossref PubMed Scopus (829) Google Scholar These editors consist of nCas9 with an inactivated HNH domain (H840A) fused to an engineered Moloney murine leukemia virus reverse transcriptase domain, making it possible to edit the genome to generate any desired sequence36Anzalone A.V. Randolph P.B. Davis J.R. Sousa A.A. Koblan L.W. Levy J.M. Chen P.J. Wilson C. Newby G.A. Raguram A. Liu D.R. Search-and-replace genome editing without double-strand breaks or donor DNA.Nature. 2019; 576: 149-157Crossref PubMed Scopus (829) Google Scholar (Figure 1D). Permanent DNA edits occur when the non-edited strand is replaced by the DNA repair system of the cell using a reverse transcriptase template containing the edit.36Anzalone A.V. Randolph P.B. Davis J.R. Sousa A.A. Koblan L.W. Levy J.M. Chen P.J. Wilson C. Newby G.A. Raguram A. Liu D.R. Search-and-replace genome editing without double-strand breaks or donor DNA.Nature. 2019; 576: 149-157Crossref PubMed Scopus (829) Google Scholar These various CRISPR-Cas systems show great potential for precise genome editing. Here, we summarized the leading strategies for CRISPR-mediated DMD gene editing below (Table 1).Table 1Summary of CRISPR-mediated therapeutic strategies to rescue the DMD phenotypeSubjectStrategyNucleaseDMD mutationTherapeutic target gene region(s)Model(s)DeliveryReferenceTherapeutic approachexon reframingSpCas9DMD exon 48–50 deletions DMD nonsense mutation in exon 51DMD exon 51human DMD myoblastselectroporation37Ousterout D.G. Kabadi A.M. Thakore P.I. Majoros W.H. Reddy T.E. Gersbach C.A. Multiplex CRISPR/Cas9-based genome editing for correction of dystrophin mutations that cause Duchenne muscular dystrophy.Nat. Commun. 2015; 6: 6244Crossref PubMed Scopus (269) Google ScholarSpCas9DMD exon 45–52 deletionsDMD exon 53human DMD myoblastsadenovirus38Maggio I. Liu J. Janssen J.M. Chen X. Gonçalves M.A.F.V. Adenoviral vectors encoding CRISPR/Cas9 multiplexes rescue dystrophin synthesis in unselected populations of DMD muscle cells.Sci. Rep. 2016; 6: 37051Crossref PubMed Scopus (31) Google ScholarSpCas9DMD exon 44 deletionDMD exon 45human iPSCselectroporation39Li H.L. Fujimoto N. Sasakawa N. Shirai S. Ohkame T. Sakuma T. Tanaka M. Amano N. Watanabe A. Sakurai H. et al.Precise correction of the dystrophin gene in duchenne muscular dystrophy patient induced pluripotent stem cells by TALEN and CRISPR-Cas9.Stem Cell Reports. 2015; 4: 143-154Abstract Full Text Full Text PDF PubMed Scopus (349) Google ScholarCjCas9Dmd nonsense mutation in exon 23Dmd exon 23DMD miceAll-in-one AAV940Koo T. Lu-Nguyen N.B. Malerba A. Kim E. Kim D. Cappellari O. Cho H.Y. Dickson G. Popplewell L. Kim J.S. Functional Rescue of Dystrophin Deficiency in Mice Caused by Frameshift Mutations Using Campylobacter jejuni Cas9.Mol. Ther. 2018; 26: 1529-1538Abstract Full Text Full Text PDF PubMed Scopus (31) Google ScholarLbCpf1DMD exon 48–50 deletions DMD nonsense mutation in exon 51DMD exon 51human iPSCsnucleofection41Zhang Y. Long C. Li H. McAnally J.R. Baskin K.K. Shelton J.M. Bassel-Duby R. Olson E.N. CRISPR-Cpf1 correction of muscular dystrophy mutations in human cardiomyocytes and mice.Sci. Adv. 2017; 3: e1602814Crossref PubMed Scopus (125) Google Scholarexon deletionSpCas9Dmd nonsense mutation in exon 23Dmd intron 22 and 23mdx miceAAV942Long C. Amoasii L. Mireault A.A. McAnally J.R. Li H. Sanchez-Ortiz E. Bhattacharyya S. Shelton J.M. Bassel-Duby R. Olson E.N. Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy.Science. 2016; 351: 400-403Crossref PubMed Scopus (582) Google ScholarSpCas9DMD exon 46–51 deletions DMD exon 46–47 deletionsDMD intron 44 and 55human DMD myoblastsnucleofection43Young C.S. Hicks M.R. Ermolova N.V. Nakano H. Jan M. Younesi S. Karumbayaram S. Kumagai-Cresse C. Wang D. Zack J.A. et al.A Single CRISPR-Cas9 Deletion Strategy that Targets the Majority of DMD Patients Restores Dystrophin Function in hiPSC-Derived Muscle Cells.Cell Stem Cell. 2016; 18: 533-540Abstract Full Text Full Text PDF PubMed Scopus (204) Google ScholarSaCas9Dmd nonsense mutation in exon 23Dmd intron 22 and 23mdx miceAAV844Nelson C.E. Hakim C.H. Ousterout D.G. Thakore P.I. Moreb E.A. Castellanos Rivera R.M. Madhavan S. Pan X. Ran F.A. Yan W.X. et al.In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy.Science. 2016; 351: 403-407Crossref PubMed Google ScholarSaCas9Dmd nonsense mutation in exon 23Dmdexon 23Ai9 mdx miceAAV945Tabebordbar M. Zhu K. Cheng J.K.W. Chew W.L. Widrick J.J. Yan W.X. Maesner C. Wu E.Y. Xiao R. Ran F.A. et al.In vivo gene editing in dystrophic mouse muscle and muscle stem cells.Science. 2016; 351: 407-411Crossref PubMed Scopus (629) Google Scholarexon skippingSpCas9DMD point mutation in intron 47, exon 51DMD exon 47A, exon 51human iPSCsnucleofection46Long C. Li H. Tiburcy M. Rodriguez-Caycedo C. Kyrychenko V. Zhou H. Zhang Y. Min Y.L. Shelton J.M. Mammen P.P.A. et al.Correction of diverse muscular dystrophy mutations in human engineered heart muscle by single-site genome editing.Sci. Adv. 2018; 4: eaap9004Crossref PubMed Scopus (104) Google ScholarSpCas9DMD exon 44 deletionDMD splice site of exon 43 or exon 45human iPSCsnucleofection47Min Y.L. Li H. Rodriguez-Caycedo C. Mireault A.A. Huang J. Shelton J.M. McAnally J.R. Amoasii L. Mammen P.P.A. Bassel-Duby R. Olson E.N. CRISPR-Cas9 corrects Duchenne muscular dystrophy exon 44 deletion mutations in mice and human cells.Sci. Adv. 2019; 5: eaav4324Crossref PubMed Scopus (84) Google ScholarSpCas9DMD nonsense mutation in exon 53DMD splice acceptor site of exon 53human DMD myoblastsadenovirus48Maggio I. Stefanucci L. Janssen J.M. Liu J. Chen X. Mouly V. Gonçalves M.A.F.V. Selection-free gene repair after adenoviral vector transduction of designer nucleases: rescue of dystrophin synthesis in DMD muscle cell populations.Nucleic Acids Res. 2016; 44: 1449-1470Crossref PubMed Scopus (47) Google ScholarSpCas9Dmd exon 50 deletionDmd splice acceptor site of exon 51DMD miceAAV949Amoasii L. Long C. Li H. Mireault A.A. Shelton J.M. Sanchez-Ortiz E. McAnally J.R. Bhattacharyya S. Schmidt F. Grimm D. et al.Single-cut genome editing restores dystrophin expression in a new mouse model of muscular dystrophy.Sci. Transl. Med. 2017; 9: eaan8081Crossref PubMed Scopus (105) Google ScholarSpCas9DMD exon 50 deletionDMD splice acceptor site of exon 51canine model of DMDAAV950Amoasii L. Hildyard J.C.W. Li H. Sanchez-Ortiz E. Mireault A. Caballero D. Harron R. Stathopoulou T.R. Massey C. Shelton J.M. et al.Gene editing restores dystrophin expression in a canine model of Duchenne muscular dystrophy.Science. 2018; 362: 86-91Crossref PubMed Scopus (218) Google Scholarhomology-directed repairSpCas9Dmd nonsense mutation in exon 23Dmd exon 23mdx miceinjection51Long C. McAnally J.R. Shelton J.M. Mireault A.A. Bassel-Duby R. Olson E.N. Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA.Science. 2014; 345: 1184-1188Crossref PubMed Scopus (436) Google ScholarSpCas9Dmd nonsense mutation in exon 23Dmd exon 23mouse muscle stem cellsadenovirus52Zhu P. Wu F. Mosenson J. Zhang H. He T.C. Wu W.S. CRISPR/Cas9-Mediated Genome Editing Corrects Dystrophin Mutation in Skeletal Muscle Stem Cells in a Mouse Model of Muscle Dystrophy.Mol. Ther. 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