Multiplex CRISPR/Cas9-based genome editing for correction of dystrophin mutations that cause Duchenne muscular dystrophy

Ousterout, David G.; Kabadi, Ami M.; Thakore, Pratiksha I.; Majoros, William H.; Reddy, Timothy E.; Gersbach, Charles A.

doi:10.1038/ncomms7244

Cited by 408 publications

(346 citation statements)

References 71 publications

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“…sgRNA and Cas9 were transferred by an adenovirus carrier into rat muscle cells; then the wrong exon was removed using the CRISPR system. These results are promising towards finding proper treatments for Duchenne muscular dystrophy in the future [26][27][28].…”

Section: Improving Muscle Function In Mice With Duchenne Muscular Dysmentioning

confidence: 97%

CRISPR genome editing and its medical applications

Mahmoudian-Sani

Farnoosh

Mahdavinezhad

et al. 2017

Biotechnology & Biotechnological Equipment

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Genome editing has always been a challenging area as a means to provide more efficient ways to create a meaningful change in the genome. Today, the CRISPR (clustered regularly interspaced short palindromic repeat) restoration system is considered as one of the suitable and promising options for genome editing. This system has many advantages compared to the previous geneediting methods developed in this area. Compared to the previous systems, CRISPR can deactivate or eliminate a gene without interfering with intracellular mechanisms. The system can be used in the treatment of diseases and in related research with identifying the performance of defective genes in these diseases. CRISPR has more potentials and applications compared to previous systems. Among these applications, we can note the use of CRISPR in understanding genetic and epigenetic diseases such as cancer. Study of cancer by the CRISPR system is done by two approaches: turning off the oncogenes and turning on the tumour suppressor genes. According to the exact capability of CRISPR, this system can also be used to create exact mutations in different cell lines to model the cancers. This type of modeling can lead to a better understanding of cancer and the ability to develop effective drugs.

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Section: Improving Muscle Function In Mice With Duchenne Muscular Dysmentioning

confidence: 97%

CRISPR genome editing and its medical applications

Mahmoudian-Sani

Farnoosh

Mahdavinezhad

et al. 2017

Biotechnology & Biotechnological Equipment

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“…The techniques described here can be extended to use multiple guide RNAs to simultaneously target several loci 14,46,47 . Many protocols have been established to differentiate hPSCs into distinct cell types, allowing various genetic manipulations in cell types of interest 30 .…”

Section: Discussionmentioning

confidence: 99%

Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation

Blair

Bateup

Hockemeyer

2016

JoVE

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Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human development and investigate the pathophysiology of disease. By employing site-specific nucleases (SSNs) for genome editing, the rapid derivation of new hPSC lines harboring specific genetic alterations in an otherwise isogenic setting becomes possible. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 are the most commonly used SSNs. All of these nucleases function by introducing a double stranded DNA break at a specified site, thereby promoting precise gene editing at a genomic locus. SSN-meditated genome editing exploits two of the cell's endogenous DNA repair mechanisms, non-homologous end joining (NHEJ) and homology directed repair (HDR), to either introduce insertion/deletion mutations or alter the genome using a homologous repair template at the site of the double stranded break. Electroporation of hPSCs is an efficient means of transfecting SSNs and repair templates that incorporate transgenes such as fluorescent reporters and antibiotic resistance cassettes. After electroporation, it is possible to isolate only those hPSCs that incorporated the repair construct by selecting for antibiotic resistance. Mechanically separating hPSC colonies and confirming proper integration at the target site through genotyping allows for the isolation of correctly targeted and genetically homogeneous cell lines. The validity of this protocol is demonstrated here by using all three SSN platforms to incorporate EGFP and a puromycin resistance construct into the AAVS1 safe harbor locus in human pluripotent stem cells.

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“…[36][37][38][39][40] For these purposes, the CRISPR/Cas9 system has been mainly exploited to remove outof-frame or in-frame mutated exons and restore the dystrophin reading-frame by targeting the sequence to be removed with two gRNAs. This approach is applicable to out-of-frame deletions and point mutations optimistically accounting for the 85% of all mutations, restoring the expression of a smaller functional protein and converting the severe phenotype into a milder, Becker-like one, as aimed by exon skipping approaches.…”

Section: Discussionmentioning

confidence: 99%

Correction of the exon 2 duplication in DMD myoblasts by a single CRISPR/Cas9 system

Lattanzi

Duguez

Moiani

et al. 2017

Neuromuscular Disorders

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Exonic duplications account for 10%-15% of all mutations in Duchenne muscular dystrophy (DMD), a severe hereditary neuromuscular disorder. We report a CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9-based strategy to correct the most frequent (exon 2) duplication in the DMD gene by targeted deletion, and tested the efficacy of such an approach in patient-derived myogenic cells. We demonstrate restoration of wild-type dystrophin expression at transcriptional and protein level in myotubes derived from genome-edited myoblasts in the absence of selection. Removal of the duplicated exon was achieved by the use of only one guide RNA (gRNA) directed against an intronic duplicated region, thereby increasing editing efficiency and reducing the risk of off-target effects. This study opens a novel therapeutic perspective for patients carrying disease-causing duplications.

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Multiplex CRISPR/Cas9-based genome editing for correction of dystrophin mutations that cause Duchenne muscular dystrophy

Cited by 408 publications

References 71 publications

CRISPR genome editing and its medical applications

CRISPR genome editing and its medical applications

Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation

Correction of the exon 2 duplication in DMD myoblasts by a single CRISPR/Cas9 system

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