CRISPR-Cas9 mediated gene-silencing of the mutant huntingtin gene in an in vitro model of huntington’s disease

Nivya Kolli, Ming Lu, Panchanan Maiti, Julien Rossignol, Gray L. Dunbar

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Huntington’s disease (HD) is a fatal neurodegenerative genetic disease characterized by a loss of neurons in the striatum. It is caused by a mutation in the Huntingtin gene (HTT) that codes for the protein huntingtin (HTT). The mutant Huntingtin gene (mHTT) contains extra poly-glutamine (CAG) repeats from which the translated mutant huntingtin proteins (mHTT) undergo inappropriate post-translational modifications, conferring a toxic gain of function, in addition to its non-functional property. In order to curb the production of the mHTT, we have constructed two CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR associate protein) plasmids, among which one nicks the DNA at untranslated region upstream to the open reading frame (uORF), and the other nicks the DNA at exon1-intron boundary. The primary goal of this study was to apply this plasmid into mesenchymal stem cells (MSCs) extracted from the bone-marrow of YAC128 mice, which carries the transgene for HD. Our results suggest that the disruption of uORF through CRISPR-Cas9 influences the translation of mHTT negatively and, to a lesser extent, disrupts the exon1-intron boundary, which affects the translation of the mHTT. These findings also revealed the pattern of the nucleotide addition or deletion at the site of the DNA-nick in this model.

Original languageEnglish
Article number754
JournalInternational Journal of Molecular Sciences
Issue number4
StatePublished - Apr 2017


  • CAG repeat
  • CRISPR-Cas9 system
  • Gene editing
  • Huntington’s disease
  • Kozak sequence
  • Mutant huntingtin
  • Pattern of NHEJ
  • YAC128


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