{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T19:19:39Z","timestamp":1791314379698,"version":"4.3.1"},"reference-count":44,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2017,4,2]],"date-time":"2017-04-02T00:00:00Z","timestamp":1491091200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Huntington\u2019s 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.<\/jats:p>","DOI":"10.3390\/ijms18040754","type":"journal-article","created":{"date-parts":[[2017,4,3]],"date-time":"2017-04-03T13:32:30Z","timestamp":1491226350000},"page":"754","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":80,"title":["CRISPR-Cas9 Mediated Gene-Silencing of the Mutant Huntingtin Gene in an In Vitro Model of Huntington\u2019s Disease"],"prefix":"10.3390","volume":"18","author":[{"given":"Nivya","family":"Kolli","sequence":"first","affiliation":[{"name":"Field Neurosciences Institute laboratory for Restorative Neurology at Central Michigan University, Mt. Pleasant, MI 48859, USA"},{"name":"Program in Neuroscience, Central Michigan University, Mt. Pleasant, MI 48859, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ming","family":"Lu","sequence":"additional","affiliation":[{"name":"Field Neurosciences Institute laboratory for Restorative Neurology at Central Michigan University, Mt. Pleasant, MI 48859, USA"},{"name":"Program in Neuroscience, Central Michigan University, Mt. Pleasant, MI 48859, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Panchanan","family":"Maiti","sequence":"additional","affiliation":[{"name":"Field Neurosciences Institute laboratory for Restorative Neurology at Central Michigan University, Mt. Pleasant, MI 48859, USA"},{"name":"Program in Neuroscience, Central Michigan University, Mt. Pleasant, MI 48859, USA"},{"name":"Department of Psychology, Central Michigan University, Mt. Pleasant, MI 48859, USA"},{"name":"Field Neurosciences Institute, St. Mary\u2019s of Michigan, Saginaw, MI 48604, USA"},{"name":"Department of Biology, Saginaw Valley State University, Saginaw, MI 48604, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Julien","family":"Rossignol","sequence":"additional","affiliation":[{"name":"Field Neurosciences Institute laboratory for Restorative Neurology at Central Michigan University, Mt. Pleasant, MI 48859, USA"},{"name":"Program in Neuroscience, Central Michigan University, Mt. Pleasant, MI 48859, USA"},{"name":"College of Medicine, Central Michigan University, Mt. Pleasant, MI 48859, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gary","family":"Dunbar","sequence":"additional","affiliation":[{"name":"Field Neurosciences Institute laboratory for Restorative Neurology at Central Michigan University, Mt. Pleasant, MI 48859, USA"},{"name":"Program in Neuroscience, Central Michigan University, Mt. Pleasant, MI 48859, USA"},{"name":"Department of Psychology, Central Michigan University, Mt. Pleasant, MI 48859, USA"},{"name":"Field Neurosciences Institute, St. Mary\u2019s of Michigan, Saginaw, MI 48604, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2017,4,2]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"496","DOI":"10.1176\/jnp.17.4.496","article-title":"Depression and stages of Huntington\u2019s disease","volume":"17","author":"Paulsen","year":"2005","journal-title":"J. Neuropsychiatry Clin. Neurosci."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"971","DOI":"10.1016\/0092-8674(93)90585-E","article-title":"A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington\u2019s disease chromosomes","volume":"72","author":"MacDonald","year":"1993","journal-title":"Cell"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"267","DOI":"10.1111\/j.1399-0004.2004.00241.x","article-title":"A new model for prediction of the age of onset and penetrance for Huntington\u2019s disease based on CAG length","volume":"65","author":"Langbehn","year":"2004","journal-title":"Clin. Genet."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"905","DOI":"10.1152\/physrev.00041.2009","article-title":"Molecular mechanisms and potential therapeutical targets in Huntington\u2019s disease","volume":"90","author":"Zuccato","year":"2010","journal-title":"Physiol. Rev."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"316","DOI":"10.1016\/j.parkreldis.2011.10.021","article-title":"An evidence-based approach in the treatment of Huntington\u2019s disease","volume":"18","author":"Mestre","year":"2012","journal-title":"Parkinsonism Relat. Disord."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Clelland, C.D., Barker, R.A., and Watts, C. (2008). Cell therapy in Huntington disease. Neurosurg. Focus, 24.","DOI":"10.3171\/FOC\/2008\/24\/3-4\/E8"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"2054","DOI":"10.1002\/stem.1135","article-title":"Neuronal properties, in vivo effects, and pathology of a Huntington\u2019s disease patient-derived induced pluripotent stem cells","volume":"30","author":"Jeon","year":"2012","journal-title":"Stem Cells"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"619","DOI":"10.1007\/s00281-011-0272-x","article-title":"To be or not to be accepted: The role of immunogenicity of neural stem cells following transplantation into the brain in animal and human studies","volume":"33","author":"Capetian","year":"2011","journal-title":"Semin. Immunopathol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"145","DOI":"10.1016\/S1474-4422(04)00678-7","article-title":"Molecular medicine for the brain: Silencing of disease genes with RNA interference","volume":"3","author":"Davidson","year":"2004","journal-title":"Lancet Neurol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1384","DOI":"10.1093\/mp\/sst075","article-title":"Zinc fingers, TAL effectors, or Cas9-based DNA binding proteins: What\u2019s best for targeting desired genome loci?","volume":"6","author":"Lahaye","year":"2013","journal-title":"Mol. Plant"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"49","DOI":"10.2174\/156652307779940216","article-title":"Meganucleases and DNA double-strand break-induced recombination: Perspectives for gene therapy","volume":"7","author":"Duchateau","year":"2007","journal-title":"Curr. Gene Ther."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"397","DOI":"10.1016\/j.tibtech.2013.04.004","article-title":"ZFN, TALEN, and CRISPR\/Cas-based methods for genome engineering","volume":"31","author":"Gaj","year":"2013","journal-title":"Trends Biotechnol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"244","DOI":"10.1046\/j.1365-2958.2000.01838.x","article-title":"Biological significance of a family of regularly spaced repeats in the genomes of Archaea, Bacteria and mitochondria","volume":"36","author":"Mojica","year":"2000","journal-title":"Mol. Microbial."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Grissa, I., Vergnaud, G., and Pourcel, C. (2007). The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats. BMC Bioinform., 8.","DOI":"10.1186\/1471-2105-8-172"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1258096","DOI":"10.1126\/science.1258096","article-title":"The new frontier of genome engineering with CRISPR-Cas9","volume":"346","author":"Doudna","year":"2014","journal-title":"Science"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"347","DOI":"10.1038\/nbt.2842","article-title":"CRISPR-Cas systems for editing, regulating and targeting genomes","volume":"32","author":"Sander","year":"2014","journal-title":"Nat. Biotechnol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"705","DOI":"10.1038\/nmeth.3015","article-title":"Microhomology-based choice of Cas9 nuclease target sites","volume":"11","author":"Bae","year":"2014","journal-title":"Nat. Methods"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"143","DOI":"10.1080\/09500340.2014.960020","article-title":"Analysis of the absorptive behavior of photopolymer materials. Part I. Theoretical modeling","volume":"62","author":"Li","year":"2015","journal-title":"J. Mod. Opt."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"4513","DOI":"10.1038\/srep04513","article-title":"Validation of microinjection methods for generating knockout mice by CRISPR\/Cas-mediated genome engineering","volume":"4","author":"Horii","year":"2014","journal-title":"Sci. Rep."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"321","DOI":"10.1038\/nrg3686","article-title":"A guide to genome engineering with programmable nucleases","volume":"15","author":"Kim","year":"2014","journal-title":"Nat. Rev. Genet."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"5110","DOI":"10.1093\/nar\/gkf664","article-title":"An upstream open reading frame impedes translation of the huntingtin gene","volume":"30","author":"Lee","year":"2002","journal-title":"Nucleic Acids Res."},{"key":"ref_22","first-page":"3438","article-title":"Effects of intercistronic length on the efficiency of reinitiation by eucaryotic ribosomes","volume":"7","author":"Kozak","year":"1987","journal-title":"Mol. Cell. Biol."},{"key":"ref_23","unstructured":"Sonenberg, N., Hershey, J., and Matthews, M. (2000). Translational control by upstream open reading frames. Translational Control of Gene Expression, Cold Spring Harbor Laboratory Press."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"8635","DOI":"10.1128\/MCB.20.23.8635-8642.2000","article-title":"Upstream open reading frames as regulators of mRNA translation","volume":"20","author":"Morris","year":"2000","journal-title":"Mol. Cell. Biol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"2281","DOI":"10.1038\/nprot.2013.143","article-title":"Genome engineering using the CRISPR-Cas9 system","volume":"8","author":"Ran","year":"2013","journal-title":"Nat. Protoc."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"12","DOI":"10.1016\/j.ymthe.2016.11.010","article-title":"CRISPR\/Cas9 editing of the mutant huntingtin allele in vitro and in vivo","volume":"25","author":"Monteys","year":"2017","journal-title":"Mol. Ther."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"224","DOI":"10.1046\/j.1365-2141.2003.04754.x","article-title":"\u03b2+45 G \u2192 C: A novel silent \u03b2-thalassaemia mutation, the first in the Kozak sequence","volume":"124","author":"Lacerra","year":"2004","journal-title":"Br. J. Haematol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"283","DOI":"10.1016\/0092-8674(86)90762-2","article-title":"Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes","volume":"44","author":"Kozak","year":"1986","journal-title":"Cell"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"461","DOI":"10.1089\/hum.2013.200","article-title":"Silencing mutant huntingtin by adeno-associated virus-mediated RNA interference ameliorates disease manifestations in the YAC128 mouse model of Huntington\u2019s disease","volume":"25","author":"Stanek","year":"2014","journal-title":"Hum. Gene Ther."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"276","DOI":"10.1002\/ana.21569","article-title":"Sustained effects of nonallele-specific Huntingtin silencing","volume":"65","author":"Drouet","year":"2009","journal-title":"Ann. Neurol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"407","DOI":"10.1126\/science.7618107","article-title":"Inactivation of the mouse Huntington\u2019s disease gene homolog Hdh","volume":"269","author":"Duyao","year":"1995","journal-title":"Science"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"155","DOI":"10.1038\/ng1095-155","article-title":"Increased apoptosis and early embryonic lethality in mice nullizygous for the Huntington\u2019s disease gene homologue","volume":"11","author":"Zeitlin","year":"1995","journal-title":"Nat. Genet."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"27","DOI":"10.1007\/BF02257483","article-title":"Structure and expression of the Huntington\u2019s disease gene: Evidence against simple inactivation due to an expanded CAG repeat","volume":"20","author":"Ambrose","year":"1994","journal-title":"Somat. Cell Mol. Genet."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"183","DOI":"10.1006\/nbdi.1996.0018","article-title":"Differential expression of normal and mutant Huntington\u2019s disease gene alleles","volume":"3","author":"Persichetti","year":"1996","journal-title":"Neurobiol. Dis."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"34","DOI":"10.1152\/nips.01410.2002","article-title":"Dysfunction of wild-type huntingtin in Huntington disease","volume":"18","author":"Cattaneo","year":"2003","journal-title":"Physiology"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"646","DOI":"10.1016\/j.bbrc.2005.02.024","article-title":"Sleeping Beauty-mediated down-regulation of huntingtin expression by RNA interference","volume":"329","author":"Chen","year":"2005","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"82","DOI":"10.1111\/j.1471-4159.2008.05734.x","article-title":"Allele-specific silencing of mutant Huntington\u2019s disease gene","volume":"108","author":"Zhang","year":"2009","journal-title":"J. Neurochem."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"17204","DOI":"10.1073\/pnas.0708285104","article-title":"Therapeutic silencing of mutant huntingtin with siRNA attenuates striatal and cortical neuropathology and behavioral deficits","volume":"104","author":"DiFiglia","year":"2007","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Shin, J.W., Kim, K.H., Chao, M.J., Atwal, R.S., Gillis, T., MacDonald, M.E., Gusella, J.F., and Lee, J.M. (2016). Permanent inactivation of Huntington\u2019s disease mutation by personalized allele-specific CRISPR\/Cas9. Hum. Mol. Genet.","DOI":"10.1093\/hmg\/ddw286"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"619","DOI":"10.1016\/j.stemcr.2017.01.022","article-title":"Reversal of Phenotypic Abnormalities by CRISPR\/Cas9-Mediated Gene Correction in Huntington Disease Patient-Derived Induced Pluripotent Stem Cells","volume":"8","author":"Xu","year":"2017","journal-title":"Stem Cell Rep."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"322","DOI":"10.1093\/nar\/gkj439","article-title":"Insights into the kinetics of siRNA-mediated gene silencing from live-cell and live-animal bioluminescent imaging","volume":"34","author":"Bartlett","year":"2006","journal-title":"Nucleic Acids Res."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"346","DOI":"10.1038\/nrg1066","article-title":"Progress and problems with the use of viral vectors for gene therapy","volume":"4","author":"Thomas","year":"2003","journal-title":"Nat. Rev. Genet."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"67","DOI":"10.1007\/978-1-61779-201-4_6","article-title":"Derivation of induced pluripotent stem cells by lentiviral transduction","volume":"767","author":"Nethercott","year":"2011","journal-title":"Methods Mol. Biol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"402","DOI":"10.1006\/meth.2001.1262","article-title":"Analysis of relative gene expression data using real-time quantitative PCR and the 2\u2212\u0394\u0394CT method","volume":"25","author":"Livak","year":"2001","journal-title":"Methods"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/18\/4\/754\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T18:31:52Z","timestamp":1760207512000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/18\/4\/754"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2017,4,2]]},"references-count":44,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2017,4]]}},"alternative-id":["ijms18040754"],"URL":"https:\/\/doi.org\/10.3390\/ijms18040754","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2017,4,2]]}}}