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. 2019 Feb 22;14(2):e0212198.
doi: 10.1371/journal.pone.0212198. eCollection 2019.

CRISPR-cas Gene-Editing as Plausible Treatment of Neuromuscular and Nucleotide-Repeat-Expansion Diseases: A Systematic Review

Free PMC article

CRISPR-cas Gene-Editing as Plausible Treatment of Neuromuscular and Nucleotide-Repeat-Expansion Diseases: A Systematic Review

Haris Babačić et al. PLoS One. .
Free PMC article


Introduction: The system of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (cas) is a new technology that allows easier manipulation of the genome. Its potential to edit genes opened a new door in treatment development for incurable neurological monogenic diseases (NMGDs). The aim of this systematic review was to summarise the findings on the current development of CRISPR-cas for therapeutic purposes in the most frequent NMGDs and provide critical assessment.

Methods and data acquisition: We searched the MEDLINE and EMBASE databases, looking for original studies on the use of CRISPR-cas to edit pathogenic variants in models of the most frequent NMGDs, until end of 2017. We included all the studies that met the following criteria: 1. Peer-reviewed study report with explicitly described experimental designs; 2. In vitro, ex vivo, or in vivo study using human or other animal biological systems (including cells, tissues, organs, organisms); 3. focusing on CRISPR as the gene-editing method of choice; and 5. featured at least one NMGD.

Results: We obtained 404 papers from MEDLINE and 513 from EMBASE. After removing the duplicates, we screened 490 papers by title and abstract and assessed them for eligibility. After reading 50 full-text papers, we finally selected 42 for the review.

Discussion: Here we give a systematic summary on the preclinical development of CRISPR-cas for therapeutic purposes in NMGDs. Furthermore, we address the clinical interpretability of the findings, giving a comprehensive overview of the current state of the art. Duchenne's muscular dystrophy (DMD) paves the way forward, with 26 out of 42 studies reporting different strategies on DMD gene editing in different models of the disease. Most of the strategies aimed for permanent exon skipping by deletion with CRISPR-cas. Successful silencing of the mHTT gene with CRISPR-cas led to successful reversal of the neurotoxic effects in the striatum of mouse models of Huntington's disease. Many other strategies have been explored, including epigenetic regulation of gene expression, in cellular and animal models of: myotonic dystrophy, Fraxile X syndrome, ataxias, and other less frequent dystrophies. Still, before even considering the clinical application of CRISPR-cas, three major bottlenecks need to be addressed: efficacy, safety, and delivery of the systems. This requires a collaborative approach in the research community, while having ethical considerations in mind.

Conflict of interest statement

The authors have declared that no competing interests exist.


Fig 1
Fig 1. Genome editing with CRISPR-cas9.
On the right—schematic depiction of genome-editing with CRISPR-cas. After delivering the CRISPR components, during the G1/S phase of the cell cycle: 1. The gRNA (DNA-binding-domain) binds the target sequence within the genome; 2. This gRNA-DNA complex is specifically recognised by the cas9 protein (the effector domain), which induces a double-stranded break in the DNA; 3. Genome modification occurs, mainly through activating one of the two DNA repair mechanisms: non-homologous end joining (NHEJ) or homology directed repair (HDR). NHEJ introduces insertions or deletions within a sequence, whereas HDR requires delivery of a donor sequence that through recombination with the targeting sequence can lead to point mutations or insertions. On the left–different delivery methods are used to deliver the CRISPR-cas9 components: viral and non-viral. Viral methods are usually more efficient but raise concerns such as immunogenicity.
Fig 2
Fig 2. PRISMA flow diagram for the systematic review.
Fig 3
Fig 3
Number of CRISPR publications: A. per year; B. per disease type. Abbreviations: DMD–Duchenne’s muscular dystrophy, HD–Huntington’s disease, DM–myotonic dystrophy, SCA–spinocerebellar ataxia, FXS–fragile X syndrome, FRDA–Friedreich’s ataxia, FSHD–facioscapulohumeral dystrophy, LGMD–limb-girdle muscular dystrophy.
Fig 4
Fig 4. Pathophysiology of the most frequent NMGDs and CRISPR-cas strategies for treatment.
A. Duchenne’s Muscular Dystrophy (DMD) is caused by mutations in the DMD gene, located on chromosome X. Mutations which lead to the formation of dysfunctional dystrophin causes the typical childhood-onset disease with severe muscular weakness and wasting, leading to death in the adolescent age. Mutations that allow the expression of a functional but shorter dystrophin cause a less severe phenotype of the disease known as Becker’s Muscular Dystrophy (BMD). Two CRISPR-cas9 strategies have been explored so far: 1. deletion of exons where the DMD mutation is located, which leads to BMD-like dystrophin expression and phenotype in mice; 2. HDR-mediated knockin that has low efficiency. B. Myotonic Dystrophy type 1 (DM1) is caused by a CTG nucleotide repeat expansion in the DMPK gene. An elongated transcribed mRNA shows toxic effects in the cells due to mis-splicing of proteins, which leads to myotonia, muscular wasting (see a schematic depiction of a typical phenotype in a boy with congenital DM1), and endocrine disorders. Authors report successful deletion of the CTG repeat expansion with CRISPR-cas9 and transcriptional downregulation of the toxic mRNA with dCas9. C. Huntington’s Disease (HD) is a CAG nucleotide repeat expansion disease. Mutation in the HTT gene leads to a systemic accumulation of an elongated HTT protein. The accumulation in the striatum of the brain gives the typical triad of symptoms: chorea, psychiatric disorders, and cognitive impairment. Studies show reversal of the neurotoxic effects after CRISPR-cas9-induced silencing of the mHTT in mice.

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