Blog
About

  • Record: found
  • Abstract: found
  • Article: found
Is Open Access

Generation of muscular dystrophy model rats with a CRISPR/Cas system

Read this article at

Bookmark
      There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

      Abstract

      Duchenne muscular dystrophy (DMD) is an X-linked lethal muscle disorder caused by mutations in the Dmd gene encoding Dystrophin 1 2. DMD model animals, such as mdx mice and canine X-linked muscular dystrophy dogs, have been widely utilized in the development of a treatment for DMD 3. Here, we demonstrate the generation of Dmd-mutated rats using a clustered interspaced short palindromic repeats (CRISPR)/Cas system, an RNA-based genome engineering technique that is also adaptive to rats. We simultaneously targeted two exons in the rat Dmd gene, which resulted in the absence of Dystrophin expression in the F0 generation. Dmd-mutated rats exhibited a decline in muscle strength, and the emergence of degenerative/regenerative phenotypes in the skeletal muscle, heart, and diaphragm. These mutations were heritable by the next generation, and F1 male rats exhibited similar phenotypes in their skeletal muscles. These model rats should prove to be useful for developing therapeutic methods to treat DMD.

      Related collections

      Most cited references 31

      • Record: found
      • Abstract: found
      • Article: not found

      Multiplex genome engineering using CRISPR/Cas systems.

      Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.
        Bookmark
        • Record: found
        • Abstract: found
        • Article: not found

        RNA-guided human genome engineering via Cas9.

        Bacteria and archaea have evolved adaptive immune defenses, termed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems, that use short RNA to direct degradation of foreign nucleic acids. Here, we engineer the type II bacterial CRISPR system to function with custom guide RNA (gRNA) in human cells. For the endogenous AAVS1 locus, we obtained targeting rates of 10 to 25% in 293T cells, 13 to 8% in K562 cells, and 2 to 4% in induced pluripotent stem cells. We show that this process relies on CRISPR components; is sequence-specific; and, upon simultaneous introduction of multiple gRNAs, can effect multiplex editing of target loci. We also compute a genome-wide resource of ~190 K unique gRNAs targeting ~40.5% of human exons. Our results establish an RNA-guided editing tool for facile, robust, and multiplexable human genome engineering.
          Bookmark
          • Record: found
          • Abstract: found
          • Article: not found

          One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering.

          Mice carrying mutations in multiple genes are traditionally generated by sequential recombination in embryonic stem cells and/or time-consuming intercrossing of mice with a single mutation. The CRISPR/Cas system has been adapted as an efficient gene-targeting technology with the potential for multiplexed genome editing. We demonstrate that CRISPR/Cas-mediated gene editing allows the simultaneous disruption of five genes (Tet1, 2, 3, Sry, Uty--8 alleles) in mouse embryonic stem (ES) cells with high efficiency. Coinjection of Cas9 mRNA and single-guide RNAs (sgRNAs) targeting Tet1 and Tet2 into zygotes generated mice with biallelic mutations in both genes with an efficiency of 80%. Finally, we show that coinjection of Cas9 mRNA/sgRNAs with mutant oligos generated precise point mutations simultaneously in two target genes. Thus, the CRISPR/Cas system allows the one-step generation of animals carrying mutations in multiple genes, an approach that will greatly accelerate the in vivo study of functionally redundant genes and of epistatic gene interactions. Copyright © 2013 Elsevier Inc. All rights reserved.
            Bookmark

            Author and article information

            Affiliations
            [1 ]Department of Veterinary Physiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo , 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
            [2 ]Department of Animal Resource Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo , 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
            [3 ]Department of Veterinary Pathology, Graduate School of Agricultural and Life Sciences, The University of Tokyo , 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
            [4 ]Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry , 4-1-1 Ogawa-Higashi, Kodaira, Tokyo 187-8502, Japan
            Author notes
            Journal
            Sci Rep
            Sci Rep
            Scientific Reports
            Nature Publishing Group
            2045-2322
            09 July 2014
            2014
            : 4
            25005781 4088098 srep05635 10.1038/srep05635
            Copyright © 2014, Macmillan Publishers Limited. All rights reserved

            This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder in order to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/

            Categories
            Article

            Uncategorized

            Comments

            Comment on this article