title: CRISPR-Cas9 Mediated Gene-Silencing of the Mutant Huntingtin Gene in an In Vitro Model of Huntington's Disease.
abstract: 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.
authors.affiliation: Field Neurosciences Institute laboratory for Restorative Neurology at Central Michigan University, Mt.Pleasant, MI 48859, USA. kolli1n@cmich.edu.|Program in Neuroscience, Central Michigan University, Mt. Pleasant, MI 48859, USA. kolli1n@cmich.edu.
authors.forename: Nivya
authors.initials: N
authors.lastname: Kolli
authors.affiliation: Field Neurosciences Institute laboratory for Restorative Neurology at Central Michigan University, Mt.Pleasant, MI 48859, USA. lu1m@cmich.edu.|Program in Neuroscience, Central Michigan University, Mt. Pleasant, MI 48859, USA. lu1m@cmich.edu.
authors.forename: Ming
authors.initials: M
authors.lastname: Lu
authors.affiliation: Field Neurosciences Institute laboratory for Restorative Neurology at Central Michigan University, Mt.Pleasant, MI 48859, USA. maiti1p@cmich.edu.|Program in Neuroscience, Central Michigan University, Mt. Pleasant, MI 48859, USA. maiti1p@cmich.edu.|Department of Psychology, Central Michigan University, Mt. Pleasant, MI 48859, USA. maiti1p@cmich.edu.|Field Neurosciences Institute, St. Mary's of Michigan, Saginaw, MI 48604, USA. maiti1p@cmich.edu.|Department of Biology, Saginaw Valley State University, Saginaw, MI 48604, USA. maiti1p@cmich.edu.
authors.forename: Panchanan
authors.initials: P
authors.lastname: Maiti
authors.affiliation: Field Neurosciences Institute laboratory for Restorative Neurology at Central Michigan University, Mt.Pleasant, MI 48859, USA. rossi1j@cmich.edu.|Program in Neuroscience, Central Michigan University, Mt. Pleasant, MI 48859, USA. rossi1j@cmich.edu.|College of Medicine, Central Michigan University, Mt. Pleasant, MI 48859, USA. rossi1j@cmich.edu.
authors.forename: Julien
authors.initials: J
authors.lastname: Rossignol
authors.affiliation: Field Neurosciences Institute laboratory for Restorative Neurology at Central Michigan University, Mt.Pleasant, MI 48859, USA. dunba1g@cmich.edu.|Program in Neuroscience, Central Michigan University, Mt. Pleasant, MI 48859, USA. dunba1g@cmich.edu.|Department of Psychology, Central Michigan University, Mt. Pleasant, MI 48859, USA. dunba1g@cmich.edu.|Field Neurosciences Institute, St. Mary's of Michigan, Saginaw, MI 48604, USA. dunba1g@cmich.edu.
authors.forename: Gray L
authors.initials: GL
authors.lastname: Dunbar
mesh_terms: D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D064113:CRISPR-Cas Systems; D002478:Cells, Cultured; D005091:Exons / Q000235:genetics; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D000071058:Huntingtin Protein / Q000235:genetics* / Q000378:metabolism; D006816:Huntington Disease / Q000235:genetics / Q000378:metabolism; D007438:Introns / Q000235:genetics; D059630:Mesenchymal Stem Cells / Q000378:metabolism; D008822:Mice, Transgenic; D008957:Models, Genetic; D050505:Mutant Proteins / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid
chemical_list: D000071058:Huntingtin Protein; D050505:Mutant Proteins
keywords: CAG repeat; CRISPR-Cas9 system; Huntington’s disease; Kozak sequence; YAC128; gene editing; mutant huntingtin; pattern of NHEJ