Splice-site defects account for about 10% of pathogenic mutations that cause Mendelian diseases1. Prevalence is higher in neuromuscular disorders (NMDs)2, owing to the unusually large size and multi-exonic nature of genes encoding muscle structural proteins. Therapeutic genome editing to correct disease-causing splice-site mutations has been accomplished only through the homology-directed repair pathway3,4,5, which is extremely inefficient in postmitotic tissues such as skeletal muscle6. Here we describe a strategy using nonhomologous end-joining (NHEJ) to correct a pathogenic splice-site mutation. As a proof of principle, we focus on congenital muscular dystrophy type 1A (MDC1A), which is characterized by severe muscle wasting and paralysis7. Specifically, we correct a splice-site mutation that causes the exclusion of exon 2 from Lama2 mRNA and the truncation of Lama2 protein in the dy2J/dy2J mouse model of MDC1A8. Through systemic delivery of adeno-associated virus (AAV) carrying clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 genome-editing components, we simultaneously excise an intronic region containing the mutation and create a functional donor splice site through NHEJ. This strategy leads to the inclusion of exon 2 in the Lama2 transcript and restoration of full-length Lama2 protein. Treated dy2J/dy2J mice display substantial improvement in muscle histopathology and function without signs of paralysis.
Subscribe to Journal
Get full journal access for 1 year
only $4.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Scotti, M.M. & Swanson, M.S. RNA mis-splicing in disease. Nat. Rev. Genet. 17, 19–32 (2016).
Bladen, C.L. et al. The TREAT-NMD DMD Global Database: analysis of more than 7,000 Duchenne muscular dystrophy mutations. Hum. Mutat. 36, 395–402 (2015).
Yang, Y. et al. A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice. Nat. Biotechnol. 34, 334–338 (2016).
Yin, H. et al. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat. Biotechnol. 32, 551–553 (2014).
Yin, H. et al. Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo. Nat. Biotechnol. 34, 328–333 (2016).
Vahidi Ferdousi, L. et al. More efficient repair of DNA double-strand breaks in skeletal muscle stem cells compared to their committed progeny. Stem Cell Res. 13 3 Pt A, 492–507 (2014).
Durbeej, M. Laminin-α2 chain-deficient congenital muscular dystrophy: pathophysiology and development of treatment. Curr. Top. Membr. 76, 31–60 (2015).
Sunada, Y., Bernier, S.M., Utani, A., Yamada, Y. & Campbell, K.P. Identification of a novel mutant transcript of laminin alpha 2 chain gene responsible for muscular dystrophy and dysmyelination in dy2J mice. Hum. Mol. Genet. 4, 1055–1061 (1995).
Geranmayeh, F. et al. Genotype-phenotype correlation in a large population of muscular dystrophy patients with LAMA2 mutations. Neuromuscul. Disord. 20, 241–250 (2010).
Guiraud, S. et al. The pathogenesis and therapy of muscular dystrophies. Annu. Rev. Genomics Hum. Genet. 16, 281–308 (2015).
Stenson, P.D. et al. The Human Gene Mutation Database: building a comprehensive mutation repository for clinical and molecular genetics, diagnostic testing and personalized genomic medicine. Hum. Genet. 133, 1–9 (2014).
Xu, H., Wu, X.R., Wewer, U.M. & Engvall, E. Murine muscular dystrophy caused by a mutation in the laminin alpha 2 (Lama2) gene. Nat. Genet. 8, 297–302 (1994).
Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).
Ran, F.A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191 (2015).
Roca, X., Krainer, A.R. & Eperon, I.C. Pick one, but be quick: 5′ splice sites and the problems of too many choices. Genes Dev. 27, 129–144 (2013).
Kuang, W. et al. Merosin-deficient congenital muscular dystrophy. Partial genetic correction in two mouse models. J. Clin. Invest. 102, 844–852 (1998).
Tanguy, Y. et al. Systemic AAVrh10 provides higher transgene expression than AAV9 in the brain and the spinal cord of neonatal mice. Front. Mol. Neurosci. 8, 36 (2015).
Zincarelli, C., Soltys, S., Rengo, G. & Rabinowitz, J.E. Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection. Mol. Ther. 16, 1073–1080 (2008).
Gombash Lampe, S.E., Kaspar, B.K. & Foust, K.D. Intravenous injections in neonatal mice. J. Vis. Exp. 93, e52037 (2014).
Yu, Q. et al. Omigapil treatment decreases fibrosis and improves respiratory rate in dy(2J) mouse model of congenital muscular dystrophy. PLoS One 8, e65468 (2013).
Tatem, K.S. et al. Behavioral and locomotor measurements using an open field activity monitoring system for skeletal muscle diseases. J. Vis. Exp. 91, 51785 (2014).
Iyer, S.R., Valencia, A.P., Hernández-Ochoa, E.O. & Lovering, R.M. In vivo assessment of muscle contractility in animal studies. Methods Mol. Biol. 1460, 293–307 (2016).
Call, J.A. & Lowe, D.A. Eccentric contraction-induced muscle injury: reproducible, quantitative, physiological models to impair skeletal muscle's capacity to generate force. Methods Mol. Biol. 1460, 3–18 (2016).
Hendel, A. et al. Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat. Biotechnol. 33, 985–989 (2015).
Tsai, S.Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat. Biotechnol. 33, 187–197 (2015).
Hirano, H. et al. Structure and engineering of Francisella novicida Cas9. Cell 164, 950–961 (2016).
Kim, E. et al. In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni. Nat. Commun. 8, 14500 (2017).
Kleinstiver, B.P. et al. Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition. Nat. Biotechnol. 33, 1293–1298 (2015).
Kleinstiver, B.P. et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature 523, 481–485 (2015).
Yeo, G. & Burge, C.B. Maximum entropy modeling of short sequence motifs with applications to RNA splicing signals. J. Comput. Biol. 11, 377–394 (2004).
Roca, X. et al. Widespread recognition of 5′ splice sites by noncanonical base-pairing to U1 snRNA involving bulged nucleotides. Genes Dev. 26, 1098–1109 (2012).
Tan, J. et al. Noncanonical registers and base pairs in human 5′ splice-site selection. Nucleic Acids Res. 44, 3908–3921 (2016).
Kemaladewi, D.U. et al. Cell-type specific regulation of myostatin signaling. FASEB J. 26, 1462–1472 (2012).
Vilquin, J.T. et al. Identification of homozygous and heterozygous dy2J mice by PCR. Neuromuscul. Disord. 10, 59–62 (2000).
Hsu, P.D., Lander, E.S. & Zhang, F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 157, 1262–1278 (2014).
Wojtal, D. et al. Spell checking nature: versatility of CRISPR/Cas9 for developing treatments for inherited disorders. Am. J. Hum. Genet. 98, 90–101 (2016).
Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).
Mali, P., Esvelt, K.M. & Church, G.M. Cas9 as a versatile tool for engineering biology. Nat. Methods 10, 957–963 (2013).
Jian, X., Boerwinkle, E. & Liu, X. In silico prediction of splice-altering single nucleotide variants in the human genome. Nucleic Acids Res. 42, 13534–13544 (2014).
LeCun, Y., Bengio, Y. & Hinton, G. Deep learning. Nature 521, 436–444 (2015).
The Cohn lab members are gratefully acknowledged for their technical support and critical input in this study. We thank I. Vukobradovic and A. Fleniken (Clinical Phenotyping Core, Toronto Centre for Phenogenomics), C. Rand (Aurora Scientific), and M. Justice, J. Dowling, and J. Ruston (Genetics and Genome Biology) for their critical inputs to this study. T. Paton, S. Perreira, G. Casallo, B. Thiruvahindrapuram, W. Sung (Toronto Center for Applied Genomics), and A. Cui (Deep Genomics) are acknowledged for their support in genomic and bioinformatics analyses. This work was supported by an AFM-Telethon postdoctoral fellowship and Cure CMD (to D.U.K.); an Ermenegildo Zegna Founder's scholarship (to E.M.); a Canada Research Chair (Tier 2) in Comparative Genomics and an Early Researcher Award from the Ontario Ministry of Research, Innovation and Science (to M.D.W.); and the Canadian Institute for Health Research, Natural Sciences and Engineering Research Council of Canada, the SickKids Foundation, RS McLaughlin Foundation and Women's Auxiliary Chairs (to R.D.C.).
The authors declare no competing financial interests.
Supplementary Tables 1–7 and Supplementary Figures 1–11 (PDF 8245 kb)
dy2J /dy2J mouse (7230) injected with combination of AAV9- Cas9-sgRNA1 and AAV9-Cas9-sgRNA2. The animal was injected at P2 via temporal vein and video was taken at the age of 10-weeks old. Hind limb paralysis, contracture and kyphosis are no longer observed, indicating improvement of the phenotypes after restoration of Lama2 protein. (MP4 11032 kb)
About this article
Cite this article
Kemaladewi, D., Maino, E., Hyatt, E. et al. Correction of a splicing defect in a mouse model of congenital muscular dystrophy type 1A using a homology-directed-repair-independent mechanism. Nat Med 23, 984–989 (2017). https://doi.org/10.1038/nm.4367
Targeted genome editing in vivo corrects a Dmd duplication restoring wild‐type dystrophin expression
EMBO Molecular Medicine (2021)
Cellular and Molecular Life Sciences (2021)
Micro-laminin gene therapy can function as an inhibitor of muscle disease in the dyW mouse model of MDC1A
Molecular Therapy - Methods & Clinical Development (2021)
Journal of Molecular Medicine (2021)
A novel mouse model of Duchenne muscular dystrophy carrying a multi-exonic Dmd deletion exhibits progressive muscular dystrophy and early-onset cardiomyopathy
Disease Models & Mechanisms (2020)