CRISPR-Cas9 Mediated Gene-Silencing of the Mutant Huntingtin Gene in an In Vitro Model of Huntington's Disease

• Published in: International journal of molecular sciences 2017-04, Vol.18 (4), p.754-754
• Main Authors: Kolli, Nivya, Lu, Ming, Maiti, Panchanan, Rossignol, Julien, Dunbar, Gray L
• Format: Article
• Language: English
• Subjects: Animals; Base Sequence; Blotting, Western; Bone marrow; Cells, Cultured; Clonal deletion; CRISPR; CRISPR-Cas Systems; Deoxyribonucleic acid; Disruption; DNA; Exons - genetics; Gene deletion; Gene Expression; Genes; Glutamine; HEK293 Cells; Humans; Huntingtin; Huntingtin Protein - genetics; Huntingtin Protein - metabolism; Huntington Disease - genetics; Huntington Disease - metabolism; Huntington's disease; Huntingtons disease; In vitro methods and tests; Introns - genetics; Mesenchymal Stromal Cells - metabolism; Mesenchyme; Mice, Transgenic; Models, Genetic; Mutant Proteins - genetics; Mutant Proteins - metabolism; Mutation; Neostriatum; Neurons; Plasmids; Polyglutamine; Post-translation; Proteins; Reverse Transcriptase Polymerase Chain Reaction; Sequence Homology, Nucleic Acid; Stem cell transplantation; Stem cells; Translation; Trinucleotide repeats
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms18040754

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 ( ) that codes for the protein huntingtin (HTT). The mutant Huntingtin gene (m ) 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.