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

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Abstract

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

Abstract

Duchenne muscular dystrophy (DMD), caused by mutations in the X-linked dystrophin gene ( DMD), is characterized by fatal degeneration of striated muscles. Dilated cardiomyopathy is one of the most common lethal features of the disease. We deployed Cpf1, a unique class 2 CRISPR (clustered regularly interspaced short palindromic repeats) effector, to correct DMD mutations in patient-derived induced pluripotent stem cells (iPSCs) and mdx mice, an animal model of DMD. Cpf1-mediated genomic editing of human iPSCs, either by skipping of an out-of-frame DMD exon or by correcting a nonsense mutation, restored dystrophin expression after differentiation to cardiomyocytes and enhanced contractile function. Similarly, pathophysiological hallmarks of muscular dystrophy were corrected in mdx mice following Cpf1-mediated germline editing. These findings are the first to show the efficiency of Cpf1-mediated correction of genetic mutations in human cells and an animal disease model and represent a significant step toward therapeutic translation of gene editing for correction of DMD.

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Most cited references 17

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Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements.

Francisco Mojica, César Díez-Villaseñor, Jesús García-Martínez … (2005)

Prokaryotes contain short DN repeats known as CRISPR, recognizable by the regular spacing existing between the recurring units. They represent the most widely distributed family of repeats among prokaryotic genomes suggesting a biological function. The origin of the intervening sequences, at present unknown, could provide clues about their biological activities. Here we show that CRISPR spacers derive from preexisting sequences, either chromosomal or within transmissible genetic elements such as bacteriophages and conjugative plasmids. Remarkably, these extrachromosomal elements fail to infect the specific spacer-carrier strain, implying a relationship between CRISPR and immunity against targeted DNA. Bacteriophages and conjugative plasmids are involved in prokaryotic population control, evolution, and pathogenicity. All these biological traits could be influenced by the presence of specific spacers. CRISPR loci can be visualized as mosaics of a repeated unit, separated by sequences at some time present elsewhere in the cell.

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Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype.

Hao Yin, Wen Xue, Sidi Chen … (2014)

We demonstrate CRISPR-Cas9-mediated correction of a Fah mutation in hepatocytes in a mouse model of the human disease hereditary tyrosinemia. Delivery of components of the CRISPR-Cas9 system by hydrodynamic injection resulted in initial expression of the wild-type Fah protein in ∼1/250 liver cells. Expansion of Fah-positive hepatocytes rescued the body weight loss phenotype. Our study indicates that CRISPR-Cas9-mediated genome editing is possible in adult animals and has potential for correction of human genetic diseases.

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Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells.

Daesik Kim, Jungeun Kim, Junho K Hur … (2016)

Programmable clustered regularly interspaced short palindromic repeats (CRISPR) Cpf1 endonucleases are single-RNA-guided (crRNA) enzymes that recognize thymidine-rich protospacer-adjacent motif (PAM) sequences and produce cohesive double-stranded breaks (DSBs). Genome editing with CRISPR-Cpf1 endonucleases could provide an alternative to CRISPR-Cas9 endonucleases, but the determinants of targeting specificity are not well understood. Using mismatched crRNAs we found that Cpf1 could tolerate single or double mismatches in the 3' PAM-distal region, but not in the 5' PAM-proximal region. Genome-wide analysis of cleavage sites in vitro for eight Cpf1 nucleases using Digenome-seq revealed that there were 6 (LbCpf1) and 12 (AsCpf1) cleavage sites per crRNA in the human genome, fewer than are present for Cas9 nucleases (>90). Most Cpf1 off-target cleavage sites did not produce mutations in cells. We found mismatches in either the 3' PAM-distal region or in the PAM sequence of 12 off-target sites that were validated in vivo. Off-target effects were completely abrogated by using preassembled, recombinant Cpf1 ribonucleoproteins.

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Author and article information

Journal

Journal ID (nlm-ta): Sci Adv

Journal ID (iso-abbrev): Sci Adv

Journal ID (publisher-id): SciAdv

Journal ID (hwp): advances

Title: Science Advances

Publisher: American Association for the Advancement of Science

ISSN (Electronic): 2375-2548

Publication date Collection: April 2017

Publication date (Electronic): 12 April 2017

Volume: 3

Issue: 4

Electronic Location Identifier: e1602814

Affiliations

[1 ]Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

[2 ]Senator Paul D. Wellstone Muscular Dystrophy Cooperative Research Center, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

[3 ]Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

[4 ]Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

Author notes

[*]

These authors contributed equally to this work.

[†]

Present address: Leon H. Charney Division of Cardiology, New York University School of Medicine, New York, NY 10016, USA.

[‡ ]Corresponding author. Email: Eric.Olson@ 123456UTSouthwestern.edu (E.N.O.); Chengzu.Long@ 123456nyumc.org (C.L.)

Author information

Kedryn K. Baskin http://orcid.org/0000-0003-3187-9655

John M. Shelton http://orcid.org/0000-0002-2949-1638

Article

Publisher ID: 1602814

DOI: 10.1126/sciadv.1602814

PMC ID: 5389745

PubMed ID: 28439558

SO-VID: f05a7145-756e-4a0a-a66c-1ca14f2ffb7b

License:

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.

History

Date received : 13 November 2016

Date accepted : 14 February 2017