CRISPR-Cas9 Mediated Gene-Silencing of the Mutant Huntingtin Gene in an In Vitro Model of Huntington’s DiseaseReportar como inadecuado


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


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1

Field Neurosciences Institute laboratory for Restorative Neurology at Central Michigan University, Mt. Pleasant, MI 48859, USA

2

Program in Neuroscience, Central Michigan University, Mt. Pleasant, MI 48859, USA

3

Department of Psychology, Central Michigan University, Mt. Pleasant, MI 48859, USA

4

Field Neurosciences Institute, St. Mary’s of Michigan, Saginaw, MI 48604, USA

5

Department of Biology, Saginaw Valley State University, Saginaw, MI 48604, USA

6

College of Medicine, Central Michigan University, Mt. Pleasant, MI 48859, USA





*

Author to whom correspondence should be addressed.



Academic Editor: Irmgard Tegeder

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. View Full-Text

Keywords: Huntington’s disease; CAG repeat; mutant huntingtin; gene editing; CRISPR-Cas9 system; pattern of NHEJ; YAC128; Kozak sequence Huntington’s disease; CAG repeat; mutant huntingtin; gene editing; CRISPR-Cas9 system; pattern of NHEJ; YAC128; Kozak sequence





Autor: Nivya Kolli 1,2, Ming Lu 1,2, Panchanan Maiti 1,2,3,4,5, Julien Rossignol 1,2,6 and Gary L. Dunbar 1,2,3,4,*

Fuente: http://mdpi.com/



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