CRISPR-Cpf1 correction of muscular dystrophy mutations in human cardiomyocytes and mice

Zhang, Yu; Long, Chengzu; Li, Hui; McAnally, John; Baskin, Kedryn K.; Shelton, John M.; Bassel‐Duby, Rhonda; Olson, Eric N.

doi:10.1126/sciadv.1602814

Cited by 214 publications

(215 citation statements)

References 33 publications

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“…Others have applied similar strategies in mdx 4cv mice, which contain a nonsense mutation in exon 53 (19). In addition, we have shown that the Cas9-related endonuclease Cpf1 can correct the Dmd mutation in mdx mice (20). …”

Section: Introductionmentioning

confidence: 99%

Single-cut genome editing restores dystrophin expression in a new mouse model of muscular dystrophy

Amoasii¹,

Long²,

Li³

et al. 2017

Sci. Transl. Med.

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217

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Duchenne muscular dystrophy (DMD) is a severe, progressive muscle disease caused by mutations in the dystrophin gene. The majority of DMD mutations are deletions that prematurely terminate the dystrophin protein. Deletions of exon 50 of the dystrophin gene are among the most common single exon deletions causing DMD. Such mutations can be corrected by skipping exon 51, thereby restoring the dystrophin reading frame. Using clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9), we generated a DMD mouse model by deleting exon 50. These ΔEx50 mice displayed severe muscle dysfunction, which was corrected by systemic delivery of adeno-associated virus encoding CRISPR/Cas9 genome editing components. We optimized the method for dystrophin reading frame correction using a single guide RNA that created reframing mutations and allowed skipping of exon 51. In conjunction with muscle-specific expression of Cas9, this approach restored up to 90% of dystrophin protein expression throughout skeletal muscles and the heart of ΔEx50 mice. This method of permanently bypassing DMD mutations using a single cut in genomic DNA represents a step toward clinical correction of DMD mutations and potentially those of other neuromuscular disorders.

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Section: Introductionmentioning

confidence: 99%

Single-cut genome editing restores dystrophin expression in a new mouse model of muscular dystrophy

Amoasii¹,

Long²,

Li³

et al. 2017

Sci. Transl. Med.

Self Cite

217

206

View full text Add to dashboard Cite

show abstract

“…The observed impairment of cell viability might be because of inhibitory effects of AsCpf1 protein itself or leaky expression of Ad proteins. 28) The former case seems less likely since any impairment of cell viability was not reported in the previous studies where Cpf1 proteins were expressed using plasmids 14,29) or AAV vectors. 14) In either case, controlling the Cpf1 expression, for example, by using the tetracyclineregulation system, or suppressing the leaky expression of Ad proteins by improved Ad vectors 30) could be the solution for this newly uncovered problem.…”

Section: Discussionmentioning

confidence: 92%

“…In fact, AsCpf1 and LbCpf1 have been shown recently to successfully perform correction of disease-related mutations in patient-derived induced pluripotent stem (iPS) cells via plasmid-electroporation as well as germline correction in the model mouse via mRNA-and gRNA-injection. 29) Taken these potential benefits, viral vector-mediated delivery of CRISPR/Cpf1 would become a promising tool for knock-in approaches in primary cells, such as hepatocytes. Our study revealed that Ad vector-mediated CRISPR/Cpf1 system could generate genome cleavages in PHHs.…”

Section: Discussionmentioning

confidence: 99%

Generation of the Adenovirus Vector-Mediated CRISPR/Cpf1 System and the Application for Primary Human Hepatocytes Prepared from Humanized Mice with Chimeric Liver

Tsukamoto

Sakai

Iizuka

et al. 2018

Biological & Pharmaceutical Bulletin

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The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) 9 system is now widely used as a genome editing tool. CRISPR-associated endonuclease in Prevotella and Francisella 1 (Cpf1) is a recently discovered Cas endonuclease that is designable and highly specific with efficiencies comparable to those of Cas9. Here we generated the adenovirus (Ad) vector carrying an Acidaminococcus sp. Cpf1 (AsCpf1) expression cassette (Ad-AsCpf1) for the first time. Ad-AsCpf1 was applied to primary human hepatocytes prepared from humanized mice with chimeric liver in combination with the Ad vector expressing the guide RNA (gRNA) directed to the Adeno-associated virus integration site 1 (AAVS1) region. The mutation rates were estimated by T7 endonuclease I assay around 12% of insertion/ deletion (indel). Furthermore, the transduced human hepatocytes were viable (ca. 60%) at two weeks post transduction. These observations suggest that the Ad vector-mediated delivery of the CRISPR/AsCpf1 system provides a useful tool for genome manipulation of human hepatocytes.

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“…The mutational hotspot has also been targeted by HDR, with mega nuclease-mediated repair of exons 45-52 in immortalized patient cells [32]. Functional dystrophin gene restoration has also been demonstrated by genome editing in iPSCs derived from a patient lacking exon 44 and 45 using TALEN and CRISPR-Cas9 [33][34][35][36] [41]. These encouraging results suggest that CRISPR/Cas9 tool possess the therapeutic potential to cure human genetic diseases including DMD.…”

Section: Dmdmentioning

confidence: 99%

Application of CRISPR/Cas9 Genome Editing in Genetic Disorders: A Systematic Review Up to Date

Pandey¹,

Tripathi²,

Bhushan³

et al. 2017

J Genet Syndr Gene Ther

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Genetic diseases in human are associated with congenital disorders and phenotypic traits. A single mutation in a gene can cause physical or mental problems, and sometimes both. Some diseases can be lethal, and there are still no cures for many of them. Socioeconomic burden of rare genetic diseases are increasing worldwide that have been tried to cure using various methods. However, they were not very successful till now. Genome editing technologies over the past few years is providing fast and effective tool to precisely manipulate the genome at specific locations. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated Cas9 (CRISPR/Cas9) system has been using from last few years in the field of biomedical research. CRISPR/Cas9 has advantages in terms of clinical applicability to treat genetic diseases like DMD, Hemophilia, β-Thalassemia and cystic fibrosis etc. and even in some cases this tool has already been successfully applied. Nevertheless, developed technologies for addition or deletion of genes have made notable progress in last few years and demonstrate some promising clinical results. However, several challenges still remain. Here, the latest applications of CRISPR-Cas9 technology in genetic disorders, current challenges and future directions are reviewed and discussed.

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CRISPR-Cpf1 correction of muscular dystrophy mutations in human cardiomyocytes and mice

Abstract: CRISPR-Cpf1–mediated correction of Duchenne muscular dystrophy mutations in human cells and a mouse model.

Cited by 214 publications

References 33 publications

Single-cut genome editing restores dystrophin expression in a new mouse model of muscular dystrophy

Single-cut genome editing restores dystrophin expression in a new mouse model of muscular dystrophy

Generation of the Adenovirus Vector-Mediated CRISPR/Cpf1 System and the Application for Primary Human Hepatocytes Prepared from Humanized Mice with Chimeric Liver

Application of CRISPR/Cas9 Genome Editing in Genetic Disorders: A Systematic Review Up to Date

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