Unexpected Mutations by CRISPR-Cas9 CTG Repeat Excision in Myotonic Dystrophy and Use of CRISPR Interference as an Alternative Approach

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

Research output: Contribution to journalArticlepeer-review

8 Citations (Scopus)


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. Ikeda et al. report unexpected mutations caused by excision of the CTG repeat in the myotonic dystrophy locus using CRISPR-Cas9 and double nicking strategy. They further show that CRISPR interference significantly suppresses the RNA foci, proposing that the alternative approach independent of double-strand breaks may be considered for therapeutic purposes.

Original languageEnglish
Pages (from-to)131-144
Number of pages14
JournalMolecular Therapy - Methods and Clinical Development
Publication statusPublished - 11-09-2020

All Science Journal Classification (ASJC) codes

  • Molecular Medicine
  • Molecular Biology
  • Genetics


Dive into the research topics of 'Unexpected Mutations by CRISPR-Cas9 CTG Repeat Excision in Myotonic Dystrophy and Use of CRISPR Interference as an Alternative Approach'. Together they form a unique fingerprint.

Cite this