Unexpected Mutations by CRISPR-Cas9 CTG Repeat Excision in Myotonic Dystrophy and Use of CRISPR Interference as an Alternative Approach
Myotonic dystrophy type 1 is the most common type of adult-onset muscular dystrophy. This is an autosomal dominant disorder and caused by the expansion of the CTG repeat in the 3′ untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. Messenger RNAs containing these expanded rep...
| Main Authors: | , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2020-09-01
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| Series: | Molecular Therapy: Methods & Clinical Development |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S232905012030111X |
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| author | Miki Ikeda Mariko Taniguchi-Ikeda Takema Kato Yasuko Shinkai Sonoko Tanaka Hiroki Hagiwara Naomichi Sasaki Toshihiro Masaki Kiichiro Matsumura Masahiro Sonoo Hiroki Kurahashi Fumiaki Saito |
| author_facet | Miki Ikeda Mariko Taniguchi-Ikeda Takema Kato Yasuko Shinkai Sonoko Tanaka Hiroki Hagiwara Naomichi Sasaki Toshihiro Masaki Kiichiro Matsumura Masahiro Sonoo Hiroki Kurahashi Fumiaki Saito |
| author_sort | Miki Ikeda |
| collection | DOAJ |
| description | Myotonic dystrophy type 1 is the most common type of adult-onset muscular dystrophy. This is an autosomal dominant disorder and caused by the expansion of the CTG repeat in the 3′ untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. Messenger RNAs containing these expanded repeats form aggregates as nuclear RNA foci. Then, RNA binding proteins, including muscleblind-like 1, are sequestered to the RNA foci, leading to systemic abnormal RNA splicing. In this study, we used CRISPR-Cas9 genome editing to excise this CTG repeat. Dual cleavage at the 5′ and 3′ regions of the repeat using a conventional Cas9 nuclease and a double nicking with Cas9 nickase successfully excised the CTG repeat. Subsequently, the formation of the RNA foci was markedly reduced in patient-derived fibroblasts. However, contrary to expectations, a considerable amount of off-target digestions and on-target genomic rearrangements were observed using high-throughput genome-wide translocation sequencing. Finally, the suppression of DMPK transcripts using CRISPR interference significantly decreased the intensity of RNA foci. Our results indicate that close attention should be paid to the unintended mutations when double-strand breaks are generated by CRISPR-Cas9 for therapeutic purposes. Alternative approaches independent of double-strand breaks, including CRISPR interference, may be considered. |
| first_indexed | 2024-12-20T15:28:40Z |
| format | Article |
| id | doaj.art-023d7e99a665465ca9bfb584a02b1aa6 |
| institution | Directory Open Access Journal |
| issn | 2329-0501 |
| language | English |
| last_indexed | 2024-12-20T15:28:40Z |
| publishDate | 2020-09-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Methods & Clinical Development |
| spelling | doaj.art-023d7e99a665465ca9bfb584a02b1aa62022-12-21T19:35:43ZengElsevierMolecular Therapy: Methods & Clinical Development2329-05012020-09-0118131144Unexpected Mutations by CRISPR-Cas9 CTG Repeat Excision in Myotonic Dystrophy and Use of CRISPR Interference as an Alternative ApproachMiki Ikeda0Mariko Taniguchi-Ikeda1Takema Kato2Yasuko Shinkai3Sonoko Tanaka4Hiroki Hagiwara5Naomichi Sasaki6Toshihiro Masaki7Kiichiro Matsumura8Masahiro Sonoo9Hiroki Kurahashi10Fumiaki Saito11Department of Neurology, School of Medicine, Teikyo University, Tokyo 1738606, JapanDepartment of Clinical Genetics, Fujita Health University Hospital, Aichi 4701192, Japan; Division of Molecular Genetics, Institute for Comprehensive Medical Science, Fujita Health University, Aichi 4701192, JapanDivision of Molecular Genetics, Institute for Comprehensive Medical Science, Fujita Health University, Aichi 4701192, JapanDivision of Molecular Genetics, Institute for Comprehensive Medical Science, Fujita Health University, Aichi 4701192, JapanDepartment of Neurology, School of Medicine, Teikyo University, Tokyo 1738606, JapanDepartment of Medical Science, Teikyo University of Science, Uenohara Campus, Yamanashi 4090193, JapanDepartment of Medical Science, Teikyo University of Science, Senju Campus, Tokyo 1200045, JapanDepartment of Medical Science, Teikyo University of Science, Senju Campus, Tokyo 1200045, JapanDepartment of Neurology, School of Medicine, Teikyo University, Tokyo 1738606, JapanDepartment of Neurology, School of Medicine, Teikyo University, Tokyo 1738606, JapanDivision of Molecular Genetics, Institute for Comprehensive Medical Science, Fujita Health University, Aichi 4701192, JapanDepartment of Neurology, School of Medicine, Teikyo University, Tokyo 1738606, Japan; Corresponding author: Fumiaki Saito, Department of Neurology, School of Medicine, Teikyo University, 2-11-1 Kaga, Itabashi-ku, Tokyo 1738606.Myotonic dystrophy type 1 is the most common type of adult-onset muscular dystrophy. This is an autosomal dominant disorder and caused by the expansion of the CTG repeat in the 3′ untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. Messenger RNAs containing these expanded repeats form aggregates as nuclear RNA foci. Then, RNA binding proteins, including muscleblind-like 1, are sequestered to the RNA foci, leading to systemic abnormal RNA splicing. In this study, we used CRISPR-Cas9 genome editing to excise this CTG repeat. Dual cleavage at the 5′ and 3′ regions of the repeat using a conventional Cas9 nuclease and a double nicking with Cas9 nickase successfully excised the CTG repeat. Subsequently, the formation of the RNA foci was markedly reduced in patient-derived fibroblasts. However, contrary to expectations, a considerable amount of off-target digestions and on-target genomic rearrangements were observed using high-throughput genome-wide translocation sequencing. Finally, the suppression of DMPK transcripts using CRISPR interference significantly decreased the intensity of RNA foci. Our results indicate that close attention should be paid to the unintended mutations when double-strand breaks are generated by CRISPR-Cas9 for therapeutic purposes. Alternative approaches independent of double-strand breaks, including CRISPR interference, may be considered.http://www.sciencedirect.com/science/article/pii/S232905012030111XCRISPR-Cas9genome editingmyotonic dystrophyDMPKRNA focioff-target |
| spellingShingle | Miki Ikeda Mariko Taniguchi-Ikeda Takema Kato Yasuko Shinkai Sonoko Tanaka Hiroki Hagiwara Naomichi Sasaki Toshihiro Masaki Kiichiro Matsumura Masahiro Sonoo Hiroki Kurahashi Fumiaki Saito Unexpected Mutations by CRISPR-Cas9 CTG Repeat Excision in Myotonic Dystrophy and Use of CRISPR Interference as an Alternative Approach Molecular Therapy: Methods & Clinical Development CRISPR-Cas9 genome editing myotonic dystrophy DMPK RNA foci off-target |
| title | Unexpected Mutations by CRISPR-Cas9 CTG Repeat Excision in Myotonic Dystrophy and Use of CRISPR Interference as an Alternative Approach |
| title_full | Unexpected Mutations by CRISPR-Cas9 CTG Repeat Excision in Myotonic Dystrophy and Use of CRISPR Interference as an Alternative Approach |
| title_fullStr | Unexpected Mutations by CRISPR-Cas9 CTG Repeat Excision in Myotonic Dystrophy and Use of CRISPR Interference as an Alternative Approach |
| title_full_unstemmed | Unexpected Mutations by CRISPR-Cas9 CTG Repeat Excision in Myotonic Dystrophy and Use of CRISPR Interference as an Alternative Approach |
| title_short | Unexpected Mutations by CRISPR-Cas9 CTG Repeat Excision in Myotonic Dystrophy and Use of CRISPR Interference as an Alternative Approach |
| title_sort | unexpected mutations by crispr cas9 ctg repeat excision in myotonic dystrophy and use of crispr interference as an alternative approach |
| topic | CRISPR-Cas9 genome editing myotonic dystrophy DMPK RNA foci off-target |
| url | http://www.sciencedirect.com/science/article/pii/S232905012030111X |
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