Your browser doesn't support javascript.
loading
Mutant Allele-Specific CRISPR Disruption in DYT1 Dystonia Fibroblasts Restores Cell Function.
Cruz, Lilian; György, Bence; Cheah, Pike See; Kleinstiver, Benjamin P; Eimer, William A; Garcia, Sara P; Sharma, Nutan; Ozelius, Laurie J; Bragg, D Cristopher; Joung, J Keith; Norberto de Souza, Osmar; Macedo Timmers, Luis Fernando Saraiva; Breakefield, Xandra O.
Afiliación
  • Cruz L; Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.
  • György B; Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA; Neurobiology Department, Harvard Medical School, Boston, MA, USA; Institute of Molecular and Clinical Ophthalmology Basel, Basel, Switzerland; Department of Ophthalmology, University of Basel, Basel, Sw
  • Cheah PS; Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA; Department of Human Anatomy, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Malaysia.
  • Kleinstiver BP; Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA; Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA; Department of Pathology, Harvard Medical School, Boston, MA, USA.
  • Eimer WA; Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA; Genetics and Aging Research Unit, MassGeneral Institute for Neurodegenerative Disease, Charlestown, MA, USA.
  • Garcia SP; Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA, USA.
  • Sharma N; Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.
  • Ozelius LJ; Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.
  • Bragg DC; Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.
  • Joung JK; Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA; Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA, USA; Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, MA, USA; Department of Pa
  • Norberto de Souza O; Laboratory for Bioinformatics, Modelling and Simulation of Biosystems-LABIO, Pontifical Catholic University of Rio Grande do Sul-PUCRS, Porto Alegre City, Brazil; Graduate Program in Cellular and Molecular Biology (PPGBCM), PUCRS, Porto Alegre - RS, Brazil.
  • Macedo Timmers LFS; University of Taquari Valley-Univates, Lajeado City, Brazil; Graduate Program in Biotechnology (PPGBiotec), Univates, Lajeado City, RS, Brazil.
  • Breakefield XO; Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA; Department of Radiology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA. Electronic address: breakefield@hms.harvard.edu.
Mol Ther Nucleic Acids ; 21: 1-12, 2020 Sep 04.
Article en En | MEDLINE | ID: mdl-32502938
Most individuals affected with DYT1 dystonia have a heterozygous 3-bp deletion in the TOR1A gene (c.907_909delGAG). The mutation appears to act through a dominant-negative mechanism compromising normal torsinA function, and it is proposed that reducing mutant torsinA may normalize torsinA activity. In this study, we used an engineered Cas9 variant from Streptococcus pyogenes (SpCas9-VRQR) to target the mutation in the TOR1A gene in order to disrupt mutant torsinA in DYT1 patient fibroblasts. Selective targeting of the DYT1 allele was highly efficient with most common non-homologous end joining (NHEJ) edits, leading to a predicted premature stop codon with loss of the torsinA C terminus (delta 302-332 aa). Structural analysis predicted a functionally inactive status of this truncated torsinA due to the loss of residues associated with ATPase activity and binding to LULL1. Immunoblotting showed a reduction of the torsinA protein level in Cas9-edited DYT1 fibroblasts, and a functional assay using HSV infection indicated a phenotypic recovery toward that observed in control fibroblasts. These findings suggest that the selective disruption of the mutant TOR1A allele using CRISPR-Cas9 inactivates mutant torsinA, allowing the remaining wild-type torsinA to exert normal function.
Palabras clave
Texto completo
Añadir a Mi BVS
Imprimir
XML
PubMed Links
Buscar en Google

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Tipo de estudio: Prognostic_studies Idioma: En Revista: Mol Ther Nucleic Acids Año: 2020 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos
Texto completo
Añadir a Mi BVS
Imprimir
XML
PubMed Links
Buscar en Google

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Tipo de estudio: Prognostic_studies Idioma: En Revista: Mol Ther Nucleic Acids Año: 2020 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos