Challenges associated with homologous directed repair using CRISPR-Cas9 and TALEN to edit the DMD genetic mutation in canine Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene that abolish the expression of dystrophin protein. Dogs with the genetic homologue, golden retriever muscular dystrophy dog (GRMD), have a splice site mutation that leads to skipping of exon 7 and a stop codon in the DMD transc...

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Veröffentlicht in:PloS one 2020-01, Vol.15 (1), p.e0228072-e0228072
Hauptverfasser: Mata López, Sara, Balog-Alvarez, Cynthia, Vitha, Stanislav, Bettis, Amanda K, Canessa, Emily H, Kornegay, Joe N, Nghiem, Peter P
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Balog-Alvarez, Cynthia
Vitha, Stanislav
Bettis, Amanda K
Canessa, Emily H
Kornegay, Joe N
Nghiem, Peter P
description Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene that abolish the expression of dystrophin protein. Dogs with the genetic homologue, golden retriever muscular dystrophy dog (GRMD), have a splice site mutation that leads to skipping of exon 7 and a stop codon in the DMD transcript. Gene editing via homology-directed repair (HDR) has been used in the mdx mouse model of DMD but not in GRMD. In this study, we used clustered regularly interspaced short palindromic repeats (CRISPR) and transcription activator-like effector nucleases (TALEN) to restore dystrophin expression via HDR in myoblasts/myotubes and later via intramuscular injection of GRMD dogs. In vitro, DNA and RNA were successfully corrected but dystrophin protein was not translated. With intramuscular injection of two different guide arms, sgRNA A and B, there was mRNA expression and Sanger sequencing confirmed inclusion of exon 7 for all treatments. On Western blot analysis, protein expression of up to 6% of normal levels was seen in two dogs injected with sgRNA B and up to 16% of normal in one dog treated with sgRNA A. TALEN did not restore any dystrophin expression. While there were no adverse effects, clear benefits were not seen on histopathologic analysis, immunofluorescence microscopy, and force measurements. Based on these results, methods must be modified to increase the efficiency of HDR-mediated gene repair and protein expression.
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Dogs with the genetic homologue, golden retriever muscular dystrophy dog (GRMD), have a splice site mutation that leads to skipping of exon 7 and a stop codon in the DMD transcript. Gene editing via homology-directed repair (HDR) has been used in the mdx mouse model of DMD but not in GRMD. In this study, we used clustered regularly interspaced short palindromic repeats (CRISPR) and transcription activator-like effector nucleases (TALEN) to restore dystrophin expression via HDR in myoblasts/myotubes and later via intramuscular injection of GRMD dogs. In vitro, DNA and RNA were successfully corrected but dystrophin protein was not translated. With intramuscular injection of two different guide arms, sgRNA A and B, there was mRNA expression and Sanger sequencing confirmed inclusion of exon 7 for all treatments. On Western blot analysis, protein expression of up to 6% of normal levels was seen in two dogs injected with sgRNA B and up to 16% of normal in one dog treated with sgRNA A. 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Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Mata López, Sara</au><au>Balog-Alvarez, Cynthia</au><au>Vitha, Stanislav</au><au>Bettis, Amanda K</au><au>Canessa, Emily H</au><au>Kornegay, Joe N</au><au>Nghiem, Peter P</au><au>Kumar, Ashok</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Challenges associated with homologous directed repair using CRISPR-Cas9 and TALEN to edit the DMD genetic mutation in canine Duchenne muscular dystrophy</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2020-01-21</date><risdate>2020</risdate><volume>15</volume><issue>1</issue><spage>e0228072</spage><epage>e0228072</epage><pages>e0228072-e0228072</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene that abolish the expression of dystrophin protein. Dogs with the genetic homologue, golden retriever muscular dystrophy dog (GRMD), have a splice site mutation that leads to skipping of exon 7 and a stop codon in the DMD transcript. Gene editing via homology-directed repair (HDR) has been used in the mdx mouse model of DMD but not in GRMD. In this study, we used clustered regularly interspaced short palindromic repeats (CRISPR) and transcription activator-like effector nucleases (TALEN) to restore dystrophin expression via HDR in myoblasts/myotubes and later via intramuscular injection of GRMD dogs. In vitro, DNA and RNA were successfully corrected but dystrophin protein was not translated. With intramuscular injection of two different guide arms, sgRNA A and B, there was mRNA expression and Sanger sequencing confirmed inclusion of exon 7 for all treatments. On Western blot analysis, protein expression of up to 6% of normal levels was seen in two dogs injected with sgRNA B and up to 16% of normal in one dog treated with sgRNA A. TALEN did not restore any dystrophin expression. While there were no adverse effects, clear benefits were not seen on histopathologic analysis, immunofluorescence microscopy, and force measurements. Based on these results, methods must be modified to increase the efficiency of HDR-mediated gene repair and protein expression.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>31961902</pmid><doi>10.1371/journal.pone.0228072</doi><tpages>e0228072</tpages><orcidid>https://orcid.org/0000-0002-0280-7217</orcidid><oa>free_for_read</oa></addata></record>
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subjects Analysis
Animals
Biology and Life Sciences
Codons
CRISPR
CRISPR-Cas Systems - genetics
Deoxyribonucleic acid
DNA
DNA binding proteins
DNA sequencing
Dogs
Duchenne muscular dystrophy
Duchenne's muscular dystrophy
Dystrophin
Dystrophin - genetics
Dystrophy
EDTA
Engineering and technology
Fluorescent antibody technique
Force measurement
Gene Editing - methods
Gene expression
Gene mutation
Gene sequencing
Genes
Genetic modification
Genetic research
Genetic Therapy - methods
Genome editing
Homology
Immunofluorescence
Injection
Medicine and Health Sciences
Messenger RNA
Microscopy
Muscular dystrophy
Muscular Dystrophy, Duchenne - genetics
Muscular Dystrophy, Duchenne - therapy
Mutation
Myoblasts
Myoblasts - cytology
Myoblasts - metabolism
Myotubes
Neomycin
Nuclease
Nucleases
Plasmids
Proteins
Repair
Research and Analysis Methods
Retirement benefits
RNA
Stop codon
Transcription
Transcription activator-like effector nucleases
Transcription Activator-Like Effector Nucleases - genetics
Utrophin
Veterinary colleges
title Challenges associated with homologous directed repair using CRISPR-Cas9 and TALEN to edit the DMD genetic mutation in canine Duchenne muscular dystrophy
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